Functional expression of yeast nitrate transporter (YNT1)and a nitrate reductase in maize

ABSTRACT

The present invention provides methods and compositions relating to altering NT activity, nitrogen utilization and/or uptake in plants. The invention relates to a method for the production of plants with maintained or increased yield under low or normal nitrogen fertility. The invention provides isolated nitrate transporter (NT) nucleic acids and their encoded proteins. The invention further provides recombinant expression cassettes, host cells, and transgenic plants. Plants transformed with nucleotide sequences encoding the NT enzyme show improved properties, for example, increased yield.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 12/860,333, filed Aug. 20, 2010, which claims the benefit U.S.Provisional Application No. 61/235,568, filed Aug. 20, 2009, both ofwhich are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The field of the disclosure relates generally to molecular biology. Inparticular, the invention relates to methods and compositions forimproving nitrogen utilization efficiency and/or nitrogen uptake inplants.

BACKGROUND OF THE INVENTION

Nitrate is the major nitrogen source for plants to uptake from soil. Tomeet the demands in the global supply of food, feed, fiber, and fuel,farmers tend to apply excessive nitrogen fertilizers to increase thegrain yield of crops, such as maize. To avoid the pollution by nitrateand reduce the cost of farming, there is a need for plants, particularlymaize, that is more efficient in nitrate uptake/utilization to maintaingrain supplies and protect our environment.

Nitrate uptake from soil into the plant root cells is an active processwhich is against an electrochemical potential gradient of the plasmamembranes. Once in the root cells, nitrate can be: 1) reduced to nitriteby the cytoplasmic enzyme nitrate reductase then ammonium by nitritereductase in chloroplasts and then incorporate into amino acids; 2)taken up and stored in vacuole; 3) transported to the xylem forlong-distance translocation into the leaves; and 4) exported out of rootcells. All steps of nitrate movements are facilitated by nitratetransporters.

Nitrate transporters can be classified into two classes based on theaffinity to nitrate, low- and high-affinity nitrate transporter systems.Low-affinity nitrate transporter systems (LATS) are responsible when thesoil nitrate concentration is higher than 1 mM and high-affinity nitratetransporter systems (HATS) play a major role when the soil nitrateconcentration is lower than 1 mM.

High-affinity nitrate transporter systems also can be classified intotwo groups based on if nitrate transporter associated protein isrequired for nitrate transporter functionality. Single-component HATScontain a protein with typical carrier-type structure with 12transmembrane domains and two-component HATS include an additional smallassociated protein with 2 transmembrance domains (Tong Y et al., PlantJ., (2005) 41:442-450). Single-component HATS involved in fungi and redalgae and two-component HATS have been reported in green algae andplants.

The expression of plant nitrate transporters can be constitutive orinduced by nitrate. Plant nitrate transporters act as component ofnitrate responsive signaling pathway and regulation of root growthindependent of nitrate uptake have be reported (Little et al., PNAS(2005) 102:13693-13698). It would be desirable to improve nitrogen useefficiency and nitrate uptake of plants; however, an attempt to improvenitrate uptake by over-expressing tobacco endogenous high affinitynitrate transporters failed. (Fraisier et al., Plant J., (2000)23:489-496).

BRIEF SUMMARY OF THE INVENTION

As described herein, a nitrate transporter (NT) from yeast Pichiaangusta, YNT1, has been shown to be involved in nitrogen uptake whenexpressed in vivo in Arabidopsis and maize plants as well as in in vitroassays. The present invention provides NT polynucleotides, codonoptimized NT gene coding sequences, related polypeptides, and allconservatively modified variants of the present NT sequences.

In another aspect, the present invention relates to a method ofincreasing yield in a plant. In one aspect, the method includesintroducing into plant cells a construct comprising a polynucleotideencoding a yeast NT such as YNT1 or a conservatively modified variant.The polynucleotide may be operably linked to a promoter functional inplant cells to yield transformed plant cells. The transformed plantcells are regenerated into a transgenic plant. The NT is expressed inthe cells of the transgenic plant at levels sufficient to increase NTactivity. In one aspect, the NT is expressed in the cells of thetransgenic plant at levels sufficient to increase plant yield.

The present invention presents methods to alter the genetic compositionof crop plants, especially maize, so that such crops can be moreproductive with current fertilizer applications and/or as productivewith significantly reduced fertilizer input. The utility of this classof invention is then both yield enhancement and reduced fertilizer costswith corresponding reduced impact to the environment.

Therefore, in one aspect, the present invention relates to an isolatednucleic acid comprising an isolated polynucleotide sequence encoding anYNT1 NT protein or a variant thereof. One embodiment of the invention isan isolated polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of: (a) the nucleotide sequence comprising SEQID NO: 3; and (b) the nucleotide sequence comprising at least 70%sequence identity to SEQ ID NO: 3 wherein said polynucleotide encodes apolypeptide having NT activity.

In another aspect, the present invention relates to a recombinantexpression cassette comprising a nucleic acid as described.Additionally, the present invention relates to a vector containing therecombinant expression cassette. Further, the vector containing therecombinant expression cassette can facilitate the transcription andtranslation of the nucleic acid in a host cell. The present inventionalso relates to the host cells able to express the NT polynucleotidesdescribed herein, including for example, YNT1 and codon optimized YNT1gene coding sequences. A number of host cells could be used, such as butnot limited to, microbial, mammalian, plant, or insect.

In yet another embodiment, the present invention is directed to atransgenic plant or plant cells, containing the nucleic acids of thepresent invention. Preferred plants containing the polynucleotides ofthe present invention include but are not limited to maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,tomato, and millet. In another embodiment, the transgenic plant is amaize plant. Another embodiment is the transgenic seeds from the plantcomprising the NT polynucleotides described herein operably linked to apromoter that drives expression in the plant. The plants of theinvention can have altered NT as compared to a control plant. In someplants, the NT is altered in a root tissue or vegetative tissue. Plantsof the invention can have at least one of the following phenotypesincluding but not limited to: increased root mass, increased rootlength, increased leaf size, increased ear size, increased seed size,increased endosperm size, and increased biomass or combinations thereof.

Another embodiment of the invention are plants that have beengenetically modified at a genomic locus, so that the genomic locusencodes a NT polypeptide encoding a YNT1 or variants thereof. Methodsfor increasing the NT activity in a plant are provided. The method cancomprise introducing into the plant an NT polynucleotide encoding aYNT1. Methods for reducing or eliminating the level of NT polypeptide inthe plant are provided.

In another aspect, the present invention relates to polynucleotidesencoding a Porphyra perforata nitrate reductase (PPNR), particularlypolynucleotides encoding a PPNR in which the alanine at amino acidposition 551 has been substituted with glycine (A551G) and a PPNR inwhich the alanine at amino acid position 551 has been substituted withglycine and the serine at amino acid position 561 has been substitutedwith aspartic acid (A551G S561D)). Such NR polynucleotides include, butare not limited to, codon optimized polynucleotides such as, forexample, a maize codon optimized polynucleotide encoding PPNR A551GS561D, which is referred to herein as PPNR A551G S561D MO. The NRpolynucleotides of the invention can be used to transform a plant byitself or stacked with one or more YNT1 polynucleotides of the inventionand used in methods to improve NUE, nitrate uptake, nitrogenassimilation, root biomass, or combinations thereof in a plant.

The present invention also provides for expression cassettes comprisingat least one YNT1 polynucleotide or NR polynucleotide of the presentinvention. In another aspect, the present invention is directed to ahost cell transfected with the recombinant expression cassettecomprising a promoter functional in a plant operably linked to any ofthe isolated polynucleotides encoding polypeptides of the presentinvention. Also provided are transformed plants, plant parts, plantcells, and seeds comprising at least one expression cassette of thepresent invention.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawing and Sequence Listing which forma part of this application.

FIG. 1 is a graphical representation of the results of field yieldtrials of maize transgenic plants expressing ZM-RM2:ADHI intron:YNT1 at3 locations under normal nitrogen (NN) conditions. Seven events weretested at 3 locations and three events were tested at 2 location. Therelative yield (%) to the null is presented here.

FIG. 2 is a graphical representation of the results of field yieldtrials of maize transgenic plants expressing ZM-RM2:ADHI intron:YNT1 at3 locations under low nitrogen (LN) conditions. Seven events were testedat 3 locations and three events were tested at 2 location. The relativeyield (%) to the null is presented here.

FIGS. 3A-3B show a nucleotide sequence comparison of YNT1 ORF (SEQ IDNO:1) and YNT1MO ORF (SEQ ID NO:3).

FIGS. 4A-4E show a nucleotide sequence comparison of wild type Porphyraperforata nitrate reductase (PPNR) ORF (SEQ ID NO:6), Porphyra perforatanitrate reductase (PPNR) A551G ORF (SEQ ID NO:4), Porphyra perforatanitrate reductase (PPNR) A551G S561D MO ORF (SEQ ID NO:8).

FIGS. 5A-5B show an amino acid sequence comparison of wild type Porphyraperforata nitrate reductase (PPNR) (SEQ ID NO:7), Porphyra perforatenitrate reductase (PPNR) A551G (SEQ ID NO:5), and Porphyra perforatenitrate reductase (PPNR) A551G S561D MO (SEQ ID NO:9).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention relates to the expression of a yeast nitratetransporter (YNT1) sequences and variants thereof in plants. While otherattempts to improve nitrate uptake in a plant by over-expressing anitrate transporter have failed (see, for example, Fraisier et al.,Plant J., (2000) 23:489-496), it is shown herein that maize transgeniclines expressing yeast nitrate transporter (YNT1) driven by aroot-preferred promoter have improved yield compared to thenon-transgenic siblings (null) under normal nitrogen yield trials in thefield and have improved yield potential compared to the null under lownitrogen conditions in greenhouse. See FIGS. 1 and 2, and Examples 9 and10 respectively. Moreover, maize plants transgenic for YNT1 also shownto have improved plant dry matters and increased nitrogen concentrationat seedling stage under low nitrogen conditions in greenhouse. SeeExample 9 below. Accordingly, plants expressing YNT1 or variants thereofas described herein may have improved nitrate uptake when grown undernormal or limited nitrogen fertility or enhance nitrogen use efficiency(NUE) of the plants.

As described elsewhere herein, the methods include expressing in a planta NT polynucleotide as described herein. Such polynucleotides includethose that encode YNT1, codon optimized YNT1 gene coding sequences, andvariants of these sequences.

For example, suitable NTs for use in the methods described hereininclude the coding portions of NT sequences which are optimized forexpression in a particular plant, such as maize. Expression may beoptimized for the specific plant by engineering the polynucleotidesequence so that it uses the plant preferred codon. As used herein, thepreferred codon refers to the preference exhibited by a specific hostcell in the usage of nucleotide codons to specify a given amino acid.The preferred codon for an amino acid for a particular host is thesingle codon which most frequently encodes that amino acid in that host.The maize preferred codon for a particular amino acid may be derivedfrom known gene sequences from maize. For example, when the plant ismaize, the preferred codon usage may be determined by evaluating knowngenes from maize plants and determining how frequently a particularcodon is used. See also Table 4 of Murray et al., Nucleic AcidsResearch, 17:477-498 (1989). For instance, the maize preferred codon foralanine is GCC, since, according to pooled sequences of 26 maize genesin Murray et al., supra, that codon encodes alanine 36% of the time,compared to GCG (24%), GCA (13%), and GCT (27%) Table 4 of Murray et al.is reproduced below.

In some cases, each codon in the NT sequence will be optimized forexpression in maize using maize preferred codons, for example, where theNT sequence of the polynucleotide comprises 100 percent of the maizepreferred codon sequences for the particular NT polypeptide. Forexample, the NT polynucleotide of SEQ ID NO:3 (YNT1MO) has a nucleotidesequence that comprises 100 percent maize preferred codon sequences andencodes a polypeptide with the same amino acid sequence as that producedby the native YNT1 protein (SEQ ID NO:2). Accordingly, the sequence ofthe NT polynucleotide is modified for optimized maize expression. Insome cases, the NT polynucleotide sequence may be modified so that theoverall G+C content of the ORF of polynucleotide is 60%, 65%, 70%, 75%,80%, 85%, 90% or more of the total length of the sequence coding for theopen-reading frame of the NT. In another aspect, the NT sequence may bemodified so that restriction sites, cryptic intron donor or acceptorsites or both, RNA instability sites, and long homogenous base stretchesor combinations thereof are eliminated.

TABLE 1 Sequences SEQUENCE SEQUENCE NAME ID NUMBER YNT1 ORF wild typepolynucleotide SEQ ID NO: 1 YNT1 wild type polypeptide SEQ ID NO: 2 YNT1maize optimized ORF polynucleotide SEQ ID NO: 3 PPNR A551G ORFpolynucleotide SEQ ID NO: 4 PPNR A551G polypeptide SEQ ID NO: 5 PPNRwild type ORF polynucleotide SEQ ID NO: 6 PPNR wild type polypeptide SEQID NO: 7 PPNR A551G maize optimized ORF polynucleotide SEQ ID NO: 8 PPNRA551G S561D maize optimized polypeptide SEQ ID NO: 9

In some aspects, the NT polynucleotide may be partially optimized forthe plant cell in which it is to be expressed. For example, when the NTpolynucleotide is to be expressed in maize, the maize NT polynucleotideis comprised of sequences or variants of the YNT1 polynucleotide whichhave been in part optimized for expression in maize. The partiallyoptimized NT polynucleotide expresses the NT protein at a levelsufficient to increase NT activity, for example, increase the yield ofthe plant, and such expression may be at a higher level than achieved ascompared to a control, e.g. a corresponding NT polynucleotide sequencewhose sequence has not been modified for expression in maize to includemaize preferred codons. Partially plant optimized sequences includethose in which, with respect to the entire length of the sequence, thesequence contains at least about 30%, 40%, 50% 60%, 70%, 80%, 90% or100% of the plant-preferred codons. Accordingly, when the partiallyplant optimized sequence is for a maize plant, the sequence may includean overall sequence that contains at least about 30%, 40%, 50% 60%, 70%,80%, 90% or 100% of the maize-preferred codons.

The NTs, including yeast NTs, specific plant optimized and partiallyoptimized NTs, may be tested for expression level of the cognate NTprotein using a transient expression assay, e.g. a maize transientexpression assay such as that described in Example 25. Using the maizeoptimized NT polynucleotides of the present invention, the level ofexpression of the NT protein may be increased at least about 2 fold, 5fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold as compared to a non-optimized NTpolynucleotide.

Modulation of the expression level of the NTs described herein wouldprovide a mechanism for manipulating a plant's nitrogen utilizationefficiency (NUE). Accordingly, the present invention provides methods,polynucleotides, and polypeptides for the production of plants withmaintained or improved yield under limited nitrogen supply or normalnitrogen conditions or both. In one aspect, the methods includeintroducing into a plant cell, plant tissue or plant one or morepolynucleotides encoding NT polypeptides described herein having theenzymatic activity of a nitrate transporter (NT). This may beaccomplished by introducing into the plant nuclear genome the nitratetransporter polynucleotides driven by a suitable promoter, for example,a constitutive promoter or a root-preferred promoter. Exemplary suitablepromoters are described elsewhere herein.

Advantageously, plants expressing NTs as described herein may providethe customer increased revenue by lowering input costs or increasingyields with a significant reduction in applied nitrogen fertilizer orboth. Furthermore, yields may be maintained or increased in plantsexpressing a NT as described herein even under non-favorable growthconditions, for example, where nitrogen is in limited supply.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Langenheim and Thimann,(1982) Botany: Plant Biology and Its Relation to Human Affairs, JohnWiley; Cell Culture and Somatic Cell Genetics of Plants, vol. 1, Vasil,ed. (1984); Stanier, et al., (1986) The Microbial World, 5^(th) ed.,Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant PathologyMethods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: ALaboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985);Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization,Hames and Higgins, eds. (1984); and the series Methods in Enzymology,Colowick and Kaplan, eds, Academic Press, Inc., San Diego, Calif.

DEFINITIONS

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole. In describing thepresent invention, the following terms will be employed, and areintended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic andprokaryotic microorganisms), such as fungi, yeast, bacteria,actinomycetes, algae and protozoa, as well as other unicellularstructures.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, Persing, et al., eds.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidsthat encode identical or conservatively modified variants of the aminoacid sequences. Because of the degeneracy of the genetic code, a largenumber of functionally identical nucleic acids encode any given protein.For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations” and represent one species ofconservatively modified variation. Every nucleic acid sequence hereinthat encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; one exception is Micrococcus rubens, for which GTGis the methionine codon (Ishizuka, et al., (1993) J. Gen. Microbiol.139:425-32) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid, which encodes apolypeptide of the present invention, is implicit in each describedpolypeptide sequence and incorporated herein by reference.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” when the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived. Forexample, substrate specificity, enzyme activity, or ligand/receptorbinding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%,preferably 60-90% of the native protein for it's native substrate.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton, Proteins, W.H. Freeman and Co. (1984).

As used herein, “consisting essentially of” means the inclusion ofadditional sequences to an object polynucleotide where the additionalsequences do not selectively hybridize, under stringent hybridizationconditions, to the same cDNA as the polynucleotide and where thehybridization conditions include a wash step in 0.1×SSC and 0.1% sodiumdodecyl sulfate at 65° C.

By “encoding” or “encoded,” with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolumn (Yamao, et al.,(1985) Proc. Natl. Acad. Sci. USA 82:2306-9), or the ciliateMacronucleus, may be used when the nucleic acid is expressed using theseorganisms.

As mentioned above, when the nucleic acid is prepared or alteredsynthetically, advantage can be taken of known codon preferences of theintended host where the nucleic acid is to be expressed. For example,host cells include but are not limited to maize, soybean, sunflower,sorghum, canola, wheat, alfalfa, cotton, rice, barley, tomato, andmillet cells. Although nucleic acid sequences of the present inventionmay be expressed in both monocotyledonous and dicotyledonous plantspecies, sequences can be modified to account for the specific codonpreferences and GC content preferences of monocotyledonous plants ordicotyledonous plants as these preferences have been shown to differ(Murray, et al., (1989) Nucleic Acids Res. 17:477-98 and hereinincorporated by reference). Thus, the maize preferred codon for aparticular amino acid might be derived from known gene sequences frommaize. Maize codon usage for 28 genes from maize plants is listed inTable 4 of Murray, et al., supra.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell, which comprises a heterologous nucleicacid sequence of the invention, which contains a vector and supports thereplication and/or expression of the expression vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, plant, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells, including but notlimited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice,cotton, canola, barley, millet, and tomato. A particularly preferredmonocotyledonous host cell is a maize host cell. In one embodiment, thehost cells are non-human host cells.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The terms “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment. Nucleicacids, which are “isolated”, as defined herein, are also referred to as“heterologous” nucleic acids. Unless otherwise stated, the term “NTnucleic acid” means a nucleic acid comprising a polynucleotide (“NTpolynucleotide”) encoding a full length or partial length NTpolypeptide.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules, which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, (1987) Guide To Molecular Cloning Techniques, from the seriesMethods in Enzymology, vol. 152, Academic Press, Inc., San Diego,Calif.; Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual,2^(nd) ed., vols. 1-3; and Current Protocols in Molecular Biology,Ausubel, et al., eds, Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (1994Supplement).

As used herein “operably linked” includes reference to a functionallinkage between a first sequence, such as a promoter, and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants, which can be used in the methodsof the invention, is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants including species from the genera: Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.Plants of the invention include, but are not limited to, rice, wheat,peanut, sugarcane, sorghum, corn, cotton, soybean, vegetable,ornamental, conifer, alfalfa, spinach, tobacco, tomato, potato,sunflower, canola, barley or millet Brassica sp., safflower, sweetpotato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea,banana, palm, avocado, fig, guava, mango, olive, papaya, cashew,macadamia, almond, sugar beet, sugarcane, buckwheat, triticale, spelt,linseed, sugar cane, oil seed rape, canola, cress, Arabidopsis,cabbages, soya, pea, beans, eggplant, bell pepper, Tagetes, lettuce,Calendula, melon, pumpkin, squash and zucchini or oat plant. Aparticularly preferred plant is Zea mays.

As used herein, “yield” may include reference to bushels per acre of agrain crop at harvest, as adjusted for grain moisture (15% typically formaize, for example), and the volume of biomass generated (for foragecrops such as alfalfa, and plant root size for multiple crops). Grainmoisture is measured in the grain at harvest. The adjusted test weightof grain is determined to be the weight in pounds per bushel, adjustedfor grain moisture level at harvest. Biomass is measured as the weightof harvestable plant material generated.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples are promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, seeds, fibres, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as “tissuepreferred.” A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “regulatable” promoter is apromoter, which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light. Anothertype of promoter is a developmentally regulated promoter, for example, apromoter that drives expression during pollen development. Tissuepreferred, cell type specific, developmentally regulated, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter, which is active under mostenvironmental conditions, for example, the ubiquitin gene promoter Ub1(GenBank accession no S94464).

As used herein, the term nitrate transporter (NT) includes but is notlimited to the sequences disclosed herein, such as NT, theirconservatively modified variants, regardless of source and any othervariants which retain the biological properties of the NT, for example,NT activity as disclosed herein. The term “NT polypeptide” refers to oneor more amino acid sequences. The term is also inclusive of fragments,variants, homologs, alleles or precursors (e.g., preproproteins orproproteins) thereof. A “NT protein” comprises a NT polypeptide. Unlessotherwise stated, the term “NT nucleic acid” means a nucleic acidcomprising a polynucleotide (“NT polynucleotide”) encoding a NTpolypeptide.

As used interchangeably herein, a “NT activity”, “biological activity ofNT” or “functional activity of NT”, refers to an activity exerted by aNT protein, polypeptide or portion thereof as determined in vivo, or invitro, according to standard techniques. In one aspect, a NT activity isthe uptake of nitrate. In one aspect, NT activity includes but is notlimited to increased nitrate specificity for nitrate, for example,decreased K_(m) for nitrate, increased velocity (V_(max)) for nitrateuptake, increased turnover rate for nitrate, and the like as compared toNT activity of an endogenous NT of a crop plant of interest. Theactivity of an NT of the present invention may be compared with anappropriate control, for example, in vitro in a yeast system expressingan individual functional plant NT or in vivo, e.g. in a plant having anNT of the invention as compared to a control plant, a plant nottransgenic for an NT of the present invention and/or transformed with anull construct. In another aspect, NT activity includes but is notlimited to increasing nitrogen use efficiency (NUE) and/or plantproductivity/yield as compared to a control plant. The NUE may inferredor determined by evaluating any number of components of NUE, includingbut not limited to remobilization of N, seed filling stage, stay green(chlorophyll content), senescence, the amount of nitrogen uptake, rateof nitrogen uptake under conditions of non-limiting or limiting Nconditions. Assays for use in determining various aspects of NUE aredescribed in the Examples herein and include but are not limited toIcoria Root NUE, NUE soil assay of Arabidopsis, TTC assay as describedin U.S. patent application Ser. No. 61/227,276, biomass evaluation, andchlorophyll content (SPAD) assays. Additional assays for measuring NUEaspects will be known to one skilled in the art.

In one aspect, the invention includes an isolated or recombinantpolypeptide with increased NT activity relative to naturally occurringenzymes involved in nitrate transport, e.g., a wild type NT enzyme.Generally, such polypeptides are NT's. For example, isolated orrecombinant polypeptides of the invention have an NT activity that is atleast about 1-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold,4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold,7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 11-fold,11.5-fold, 12.0-fold, 12.5-fold, 13-fold, 13.5-fold, 14.0-fold,14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold,17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold,21-fold, 21.5-fold, 22.0-fold, 22.5-fold, 23-fold, 23.5-fold, 24.0-fold,24.5-fold, 25.0-fold, 25.5-fold, 26.0-fold, 26.5-fold, 27.0-fold,27.5-fold, 28.0-fold, 28.5-fold, 29.0-fold, 29.5-fold, 30.0-fold, orgreater than a naturally occurring (native or wild-type) enzyme, such asexemplified by SEQ ID NO:2 of YNY1 NT or as described elsewhere herein.

The expression level of the NT polypeptide may be measured directly, forexample, by measuring the level of the NT polypeptide by Western in theplant, or indirectly, for example, by measuring the NT activity of theNT polypeptide in the plant. Methods for determining the NT activity maybe determined using known methods such as mutant complementation with aknown NT, including the evaluation of the expression of the putautive NTgene or activity in various expression systems, for example of Xenopusoocytes (See Miller, A. J. and Zhou, J. J., Xenopus Oocytes as anExpression System for Plant Transporters, Biochimica et Biophysica Acta.(2000). 1465: 343-358) or in a yeast system of Pichia pastoris describedin U.S. patent application Ser. No. 12/136,173. See, for example,Accumulation of nitrate in the shoot acts as a signal to regulate shoot:root allocation in tobacco. Plant J. 11: 671-691. See also, for example,a pH dye based system for measuring nitrate uptake in patent applicationpublication Ser. No. 12/166,473, U.S. Patent Application Publication No.2009/0011516. Methods for determining the reduction of nitrate tonitrite, nitrate reduction rate and/or specificity for nitrate, may bedetermined using standard techniques such as a Griess reactioncolorimetric assay and those described in Hageman et al., MethodsEnzymol. (1971) 23:491-503, Tucker D E, Allen D J Ort D R (2004).Control of nitrate reductase by circadium and diurnal rhythms in tomato.Planta 219:277-285. and Scheible W R, Lauerer M, Schultze E D, CabocheM, Stitt M (1997), Fiddler R M, Collaborative Study of Modified AOACMethod of Analysis for Nitrite in Meat and Meat Products, J. AOAC, 60,594-99, (1977).

NT activity may also include evaluation of phenotypic changes, such asincreased or maintained yield or NUE in a plant grown under nitratelimiting conditions such as lower nitrogen fertility. Examples ofphenotypic changes include but are not limited to increased ear size inmaize, increased shoot biomass, increased ear growth rate, increasedbiomass, higher grain yields, synchronous flowering so that pollen isshed at approximately the same time as silking, enhanced root growth,enhanced root structures, increased seed size, increased seed weight,seed with increased embryo size, increased leaf size, increased seedlingvigor, enhanced silk emergence, and greater chlorophyll content(greener).

Maintained or increased yield may be achieved through the NTs describedherein. Thus, modulation of NT activity in a plant cell using the NT'sdescribed herein provides a novel strategy for maintaining or increasingyield or NUE of a plant grown under limited nitrogen supply or lowernitrogen fertility

Accordingly, the present invention further provides plants havingincreased yield or a maintained yield when grown under limited nitrogenfertility. In some embodiments, the plants having an increased ormaintained yield when grown under limited nitrogen fertility have amodulated level of NT or NT activity or both.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been affected as to a gene of interest, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A “control plant or plant cell” may comprise, for example: (a) awild-type plant or cell, i.e., of the same genotype as the startingmaterial for the genetic alteration which resulted in the subject plantor cell; (b) a plant or plant cell of the same genotype as the startingmaterial but which has been transformed with a null construct (i.e. witha construct which has no known effect on the trait of interest, such asa construct comprising a marker gene); (c) a plant or plant cell whichis a non-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all as a result of deliberate human intervention; ormay have reduced or eliminated expression of a native gene. The term“recombinant” as used herein does not encompass the alteration of thecell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant polynucleotide” is a polynucleotide thatis produced by recombinant DNA technology such as, for example, theshuffled polynucleotides disclosed herein. A “recombinant polypeptide”is a polypeptide encoded by a recombinant polynucleotide. Preferrably,the recombinant polynucleotides of the invention do not have the samenucleotide sequence as that of a naturally occurring polynucleotide.Preferrably, the recombinant polypeptides of the invention do not havethe same amino acid sequence as that of a naturally occurringpolypeptide.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements, which permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 40% sequence identity, preferably 60-90% sequenceidentity, and most preferably 100% sequence identity (i.e.,complementary) with each other.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which can be up to 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Optimally, the probe is approximately 500 nucleotides inlength, but can vary greatly in length from less than 500 nucleotides toequal to the entire length of the target sequence.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide or Denhardt's.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-84:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995). Unless otherwise stated, in thepresent application high stringency is defined as hybridization in4×SSC, 5×Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovineserum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA,and 25 mM Na phosphate at 65° C., and a wash in 0.1×SSC, 0.1% SDS at 65°C.

As used herein, “transgenic plant” includes reference to a plant, whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

“Variants” is intended to include substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the parentalpolynucleotide, e.g. a native polynucleotide from a fungus or plant,that may be codon-optized, and/or a substitution of one or morenucleotides at one or more sites in the parental polynucleotide. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the NT polypeptides of the invention. Naturallyoccurring variants such as these can be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode an NT protein employedin the invention. Generally, variants of a particular polynucleotide ofthe invention will have at least about 50%, 55%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to a particular reference polynucleotide, e.g.,native NT polynucleotide or template NT polynucleotide, as determined bysequence alignment programs and parameters described elsewhere herein.Accordingly, NT polynucleotides that have 50%, 55%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to a polynucleotide of SEQ ID NO: 3, 5, 7, 9, or11 are contemplated.

Variants of a particular polynucleotide employed in the invention (i.e.,the reference or parental polynucleotide) can also be evaluated bycomparison of the sequence identity between the polypeptide encoded by avariant polynucleotide and the polypeptide encoded by the reference orparental polynucleotide. Thus, for example, an isolated polynucleotidethat encodes a polypeptide with a given percent sequence identity to anyone of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, or 10 is encompassed.Percent sequence identity between any two polypeptides can be calculatedusing sequence alignment programs and parameters described elsewhereherein. Where any given pair of polynucleotides of the invention isevaluated by comparison of the percent sequence identity shared by thetwo polypeptides they encode, the percent sequence identity between thetwo encoded polypeptides is at least about 50%, 55%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% ormore sequence identity.

“Variant” protein is intended to include a protein derived from thenative or parental protein by deletion, substitution or addition of oneor more amino acids at one or more sites in the native or parentalprotein and/or substitution of one or more amino acids at one or moresites in the native or parental protein. Variant proteins encompassed bythe present invention are biologically active, that is they continue topossess the desired biological activity of the native or parentalprotein, that is, NT activity as described herein. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active NTs of the invention will have atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,95%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acidsequence for the variant protein as determined by sequence alignmentprograms and parameters described elsewhere herein. Encompassed hereinare NT polypeptides that have 50%, 55%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or moresequence identity to a polypeptide of SEQ ID NO: 4, 6, 8, or 10.

A biologically active variant of a protein of the invention may differfrom that protein by 50 or more amino acid residues, 30-50 residues,15-30 amino acid residues, as few as 1-15 amino acid residues, as few as1-10, such as 6-10, as few as 5, as few as 5, 3, 2, or even 1 amino acidresidue. As used herein, the term “nitrate transporter” or “NT” includesbut is not limited to the sequences or polymorphisms disclosed herein,their conservatively modified variants, regardless of source and anyother variants which retain or increase the biological properties of theNT, for example, NT activity as disclosed herein.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides or polypeptides:(a) “reference sequence,” (b) “comparison window,” (c) “sequenceidentity,” (d) “percentage of sequence identity,” and (e) “substantialidentity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100 or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence a gap penalty is typically introduced andis subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, mayconduct optimal alignment of sequences for comparison; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG® programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTALprogram is well described by Higgins and Sharp, (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65, and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. The preferred program to use for optimal global alignment ofmultiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol.,25:351-60 which is similar to the method described by Higgins and Sharp,(1989) CABIOS 5:151-53 and hereby incorporated by reference). The BLASTfamily of programs which can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTX for nucleotide query sequences againstprotein database sequences; BLASTP for protein query sequences againstprotein database sequences; TBLASTN for protein query sequences againstnucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel et al., eds., GreenePublishing and Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch, supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the Wisconsin Genetics Software Package are 8 and 2, respectively.The gap creation and gap extension penalties can be expressed as aninteger selected from the group of integers consisting of from 0 to 100.Thus, for example, the gap creation and gap extension penalties can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl.Acad. Sci. USA 89:10915).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences, which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences, which differ by suchconservative substitutions, are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has between 50-100% sequenceidentity, preferably at least 50% sequence identity, preferably at least60% sequence identity, preferably at least 70%, more preferably at least80%, more preferably at least 90%, and most preferably at least 95%,compared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of between 55-100%,preferably at least 55%, preferably at least 60%, more preferably atleast 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.The degeneracy of the genetic code allows for many amino acidssubstitutions that lead to variety in the nucleotide sequence that codefor the same amino acid, hence it is possible that the DNA sequencecould code for the same polypeptide but not hybridize to each otherunder stringent conditions. This may occur, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide, which the first nucleicacid encodes, is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The terms “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with between 55-100% sequenceidentity to a reference sequence preferably at least 55% sequenceidentity, preferably 60% preferably 70%, more preferably 80%, mostpreferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch, supra. An indication that two peptide sequencesare substantially identical is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially identical to a second peptide, for example,where the two peptides differ only by a conservative substitution. Inaddition, a peptide can be substantially identical to a second peptidewhen they differ by a non-conservative change if the epitope that theantibody recognizes is substantially identical. Peptides, which are“substantially similar” share sequences as, noted above except thatresidue positions, which are not identical, may differ by conservativeamino acid changes.

Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids ofRNA, DNA, and analogs and/or chimeras thereof, comprising a NTpolynucleotide.

The present invention also includes polynucleotides optimized forexpression in different organisms. For example, for expression of thepolynucleotide in a maize plant, the sequence can be altered to accountfor specific codon preferences and to alter GC content as according toMurray, et al, supra. Maize codon usage for 28 genes from maize plantsis listed in Table 4 of Murray, et al., supra.

The NT nucleic acids of the present invention comprise isolated NTpolynucleotides which are inclusive of: (a) a polynucleotide encoding aNT polypeptide and conservatively modified and polymorphic variantsthereof, (b) a polynucleotide having at least 70% sequence identity withpolynucleotides of (a) or (b); (c) a complementary sequences ofpolynucleotides of (a) or (b).

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a fungus or bacteria.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the polynucleotide sequence—isoptionally a vector, adapter, or linker for cloning and/or expression ofa polynucleotide of the present invention. Additional sequences may beadded to such cloning and/or expression sequences to optimize theirfunction in cloning and/or expression, to aid in isolation of thepolynucleotide, or to improve the introduction of the polynucleotideinto a cell. Typically, the length of a nucleic acid of the presentinvention less the length of its polynucleotide of the present inventionis less than 20 kilobase pairs, often less than 15 kb, and frequentlyless than 10 kb. Use of cloning vectors, expression vectors, adapters,and linkers is well known in the art. Exemplary nucleic acids includesuch vectors as: M13, lambda ZAP Express, lambda ZAP II, lambda gt10,lambda gt11, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambdaEMBL 3, lambda EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/−,pSG5, pBK, pCR-Script, pET, pSPUTK, p3′SS, pGEM, pSK+/−, pGEX, pSPORTIand II, pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo,pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406,pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambda MOSElox.Optional vectors for the present invention, include but are not limitedto, lambda ZAP II, and pGEX. For a description of various nucleic acidssee, e.g., Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (LaJolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (ArlingtonHeights, Ill.).

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68:90-9; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-51;the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra.Letts. 22(20):1859-62; the solid phase phosphoramidite triester methoddescribed by Beaucage, et al., supra, e.g., using an automatedsynthesizer, e.g., as described in Needham-VanDevanter, et al., (1984)Nucleic Acids Res. 12:6159-68; and, the solid support method of U.S.Pat. No. 4,458,066. Chemical synthesis generally produces a singlestranded oligonucleotide. This may be converted into double stranded DNAby hybridization with a complementary sequence or by polymerization witha DNA polymerase using the single strand as a template. One of skillwill recognize that while chemical synthesis of DNA is limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 5<G> 7 methyl GpppG RNA cap structure (Drummond, etal., (1985) Nucleic Acids Res. 13:7375). Negative elements includestable intramolecular 5′ UTR stem-loop structures (Muesing, et al.,(1987) Cell 48:691) and AUG sequences or short open reading framespreceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al.,(1988) Mol. and Cell. Biol. 8:284). Accordingly, the present inventionprovides 5′ and/or 3′ UTR regions for modulation of translation ofheterologous coding sequences.

As mentioned above, the polypeptide-encoding segments of thepolynucleotides of the present invention can be modified to alter codonusage. Altered codon usage can be employed to alter translationalefficiency and/or to optimize the coding sequence for expression in adesired host or to optimize the codon usage in a heterologous sequencefor expression in maize. Codon usage in the coding regions of thepolynucleotides of the present invention can be analyzed statisticallyusing commercially available software packages such as “CodonPreference” available from the University of Wisconsin Genetics ComputerGroup. See, Devereaux, et al., (1984) Nucleic Acids Res. 12:387-395); orMacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the presentinvention provides a codon usage frequency characteristic of the codingregion of at least one of the polynucleotides of the present invention.The number of polynucleotides (3 nucleotides per amino acid) that can beused to determine a codon usage frequency can be any integer from 3 tothe number of polynucleotides of the present invention as providedherein. Optionally, the polynucleotides will be full-length sequences.An exemplary number of sequences for statistical analysis can be atleast 1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT publication No.96/19256. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-9; and Zhao, et al., (1998) Nature Biotech 16:258-61. Generally,sequence shuffling provides a means for generating libraries ofpolynucleotides having a desired characteristic, which can be selectedor screened for. Libraries of recombinant polynucleotides are generatedfrom a population of related sequence polynucleotides, which comprisesequence regions, which have substantial sequence identity and can behomologously recombined in vitro or in vivo. The population ofsequence-recombined polynucleotides comprises a subpopulation ofpolynucleotides which possess desired or advantageous characteristicsand which can be selected by a suitable selection or screening method.The characteristics can be any property or attribute capable of beingselected for or detected in a screening system, and may includeproperties of: an encoded protein, a transcriptional element, a sequencecontrolling transcription, RNA processing, RNA stability, chromatinconformation, translation, or other expression property of a gene ortransgene, a replicative element, a protein-binding element, or thelike, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be analtered K_(m) and/or K_(cat) over the wild-type protein as providedherein. In other embodiments, a protein or polynucleotide generated fromsequence shuffling will have a ligand binding affinity greater than thenon-shuffled wild-type polynucleotide. In yet other embodiments, aprotein or polynucleotide generated from sequence shuffling will have analtered pH optimum as compared to the non-shuffled wild-typepolynucleotide. The increase in such properties can be at least 110%,120%, 130%, 140% or greater than 150% of the wild-type value.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polynucleotide of the present invention,for example a cDNA or a genomic sequence encoding a polypeptide longenough to code for an active protein of the present invention, can beused to construct a recombinant expression cassette which can beintroduced into the desired host cell. A recombinant expression cassettewill typically comprise a polynucleotide of the present inventionoperably linked to transcriptional initiation regulatory sequences whichwill direct the transcription of the polynucleotide in the intended hostcell, such as tissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

A number of promoters can be used in the practice of the invention,including the native promoter of the endogenous NT polynucleotidesequence of the crop plant of interest. The promoters can be selectedbased on the desired outcome. The nucleic acids can be combined withconstitutive, tissue-preferred, inducible, or other promoters forexpression in plants.

A plant promoter or promoter fragment can be employed which will directexpression of a polynucleotide of the present invention in all tissuesof a regenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the rubisco promoter, the GRP1-8 promoter, the 35S promoterfrom cauliflower mosaic virus (CaMV), as described in Odell, et al.,(1985) Nature 313:810-2; rice actin (McElroy, et al., (1990) Plant Cell163-171); ubiquitin (Christensen, et al., (1992) Plant Mol. Biol.12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-89);pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,et al., (1984) EMBO J. 3:2723-30); and maize H3 histone (Lepetit, etal., (1992) Mol. Gen. Genet. 231:276-85; and Atanassvoa, et al., (1992)Plant Journal 2(3):291-300); ALS promoter, as described in PCTApplication No. WO 96/30530; and other transcription initiation regionsfrom various plant genes known to those of skill. For the presentinvention ubiquitin is the preferred promoter for expression in monocotplants.

Tissue-preferred promoters can be utilized to target enhanced type A RRexpression within a particular plant tissue. By “tissue-preferred” isintended to mean that expression is predominately in a particulartissue, albeit not necessarily exclusively in that tissue.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 255(3):337-353; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1351; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-525;Yamamoto et al. (1995) Plant Cell Physiol. 35(5):773-778; Lam (1995)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.5(3):595-505. Such promoters can be modified, if necessary, for weakexpression. See, also, U.S. Patent Application No. 2003/0074698, hereinincorporated by reference.

A mesophyllic cell preferred promoter includes but is not limited topromoters such as known phosphoenopyruvate decarboxylase (PEPC)promoters or putative PEPC promoters from any number of species, forexample, Zea mays, Oryza sativa, Arabidopsis thaliana, Glycine max, orSorghum bicolor. Examples include Zea mays PEPC of GenBank accession no.gi:116268332_HTG AC190686, (FIG. 12) and gCAT GSS composite sequence(FIG. 17); Oryza sativa PEPC of GenBank accession no.gi|20804452|dbj|AP003052.3| (FIG. 13); Arabidopsis thaliana PEPC ofGenBank accession nos. gi|5541653|dbj|AP000370.1|AP000370 (FIG. 14);gi:7769847 (FIG. 15); or gi|20198070|gb|AC007087.7 (FIG. 16); Glycinemax (GSS contigs) (FIGS. 18-19); or Sorghum bicolor (JGI assemblyscaffold_(—)832, 89230 bp., JGI assembly scaffold_(—)1632, FIGS. 20-21).(1997) Plant J. 12(2):255-265; Kwon et al. (1995) Plant Physiol.105:357-67; Yamamoto et al. (1995) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; Baszczynski et al. (1988) Nucl. Acid Res.16:5732; Mitra et al. (1995) Plant Molecular Biology 26:35-93; Kayaya etal. (1995) Molecular and General Genetics 258:668-675; and Matsuoka etal. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. Senescenceregulated promoters are also of use, such as, SAM22 (Crowell et al.(1992) Plant Mol. Biol. 18:559-566). See also U.S. Pat. No. 5,589,052,herein incorporated by reference.

Shoot-preferred promoters include, shoot meristem-preferred promoterssuch as promoters disclosed in Weigal et al. (1992) Cell 69:853-859;Accession No. AJ131822; Accession No. Z71981; Accession No. AF059870,the ZAP promoter (U.S. patent application Ser. No. 10/387,937), themaize tb1 promoter (Wang et al. (1999) Nature 398:236-239, andshoot-preferred promoters disclosed in McAvoy et al. (2003) Acta Hort.(ISHS) 625:379-385.

Root-preferred or root cell specific promoters are known and can beselected from the many available from the literature or isolated de novofrom various compatible species. Exemplary root-preferred promotersinclude but are not limited to root-preferred promoter, e.g. maize rootmetallothionein promoter (ZM-RM2 PRO), maize NAS2 promoter, and viralpromoters such as banana streak virus promoter truncated version (BSV(TR) PRO) and full version (BSV (FL) PRO). See U.S. Pat. No. 7,214,855issued May 8, 2007 for ZM-RM2 promoter, and U.S. patent application Ser.No. 61/184,043 filed Jun. 4, 2009, for BSV TR (BSV truncated promoter),incorporated herein in their entirety. See also, for example, Hire etal. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specificglutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell3(10):1051-1061 (root-specific control element in the GRP 1.8 gene ofFrench bean); Sanger et al. (1990) Plant Mol. Biol. 15(3):533-553(root-specific promoter of the mannopine synthase (MAS) gene ofAgrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22(full-length cDNA clone encoding cytosolic glutamine synthetase (GS),which is expressed in roots and root nodules of soybean). See alsoBogusz et al. (1990) Plant Cell 2(7):633-651, where two root-specificpromoters isolated from hemoglobin genes from the nitrogen-fixingnonlegume Parasponia andersonii and the related non-nitrogen-fixingnonlegume Trema tomentosa are described. The promoters of these geneswere linked to a β-glucuronidase reporter gene and introduced into boththe nonlegume Nicotiana tabacum and the legume Lotus corniculatus, andin both instances root-specific promoter activity was preserved. Leachand Aoyagi (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see Plant Science (Limerick) 79(1):69-76). They concluded that enhancerand tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri et al. (1989) used gene fusion to lacZ to show that theAgrobacterium T-DNA gene encoding octopine synthase is especially activein the epidermis of the root tip and that the TR2′ gene is root specificin the intact plant and stimulated by wounding in leaf tissue, anespecially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see EMBO J. 8(2):353-350). The TR1′gene, fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(5):759-772); rolB promoter (Capana et al. (1995) Plant Mol. Biol.25(5):681-691; and the CRWAQ81 root-preferred promoter with the ADHfirst intron (U.S. Pat. No. 7,411,112, filed Oct. 9, 2003, hereinincorporated by reference). See also U.S. Pat. Nos. 5,837,876;5,750,386; 5,633,363; 5,559,252; 5,501,836; 5,110,732; and 5,023,179.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adh1 promoter, whichis inducible by hypoxia or cold stress, the Hsp70 promoter, which isinducible by heat stress, and the PPDK promoter, which is inducible bylight.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds, or flowers. The operation of a promotermay also vary depending on its location in the genome. Thus, aninducible promoter may become fully or partially constitutive in certainlocations.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from a varietyof plant genes, or from T-DNA. The 3′ end sequence to be added can bederived from, for example, the nopaline synthase or octopine synthasegenes, or alternatively from another plant gene, or less preferably fromany other eukaryotic gene. Examples of such regulatory elements include,but are not limited to, 3′ termination and/or polyadenylation regionssuch as those of the Agrobacterium tumefaciens nopaline synthase (nos)gene (Bevan, et al., (1983) Nucleic Acids Res. 12:369-85); the potatoproteinase inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic AcidsRes. 14:5641-50; and An, et al., (1989) Plant Cell 1:115-22); and theCaMV 19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold (Buchman and Berg, (1988)Mol. Cell Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev.1:1183-200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adh1-S intron 1, 2 and 6, the Bronze-1 intron are known inthe art. See generally, The Maize Handbook, Chapter 116, Freeling andWalbot, eds., Springer, N.Y. (1994).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos, et al., (1989) J. Biol. Chem.264:4896-900), such as the Nicotiana plumbaginifolia extension gene(DeLoose, et al., (1991) Gene 99:95-100); signal peptides which targetproteins to the vacuole, such as the sweet potato sporamin gene(Matsuka, et al., (1991) Proc. Natl. Acad. Sci. USA 88:834) and thebarley lectin gene (Wilkins, et al., (1990) Plant Cell, 2:301-13);signal peptides which cause proteins to be secreted, such as that ofPRIb (Lind, et al., (1992) Plant Mol. Biol. 18:47-53) or the barleyalpha amylase (BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol.12:119, and hereby incorporated by reference), or signal peptides whichtarget proteins to the plastids such as that of rapeseed enoyl-Acpreductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202) areuseful in the invention.

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene, which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, and the ALS gene encodesresistance to the herbicide chlorsulfuron. Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as red fluorescent protein (RFP), green fluorescentprotein (GFP) (Su et al. (2005) Biotechnol Bioeng 85:610-9 and Fetter etal. (2005) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolteet al. (2005) J. Cell Science 117:953-55 and Kato et al. (2002) PlantPhysiol 129:913-52), and yellow florescent protein (PhiYFP™ fromEvrogen, see, Bolte et al. (2005) J. Cell Science 117:953-55).

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al.(1987), Meth. Enzymol. 153:253-77. These vectors are plant integratingvectors in that on transformation, the vectors integrate a portion ofvector DNA into the genome of the host plant. Exemplary A. tumefaciensvectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,(1987) Gene 61:1-11, and Berger, et al., (1989) Proc. Natl. Acad. Sci.USA, 86:8402-6. Another useful vector herein is plasmid pBI101.2 that isavailable from CLONTECH Laboratories, Inc. (Palo Alto, Calif.).

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian, or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or inducible), followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a strong promoter, such as ubiquitin, to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. Constitutive promoters areclassified as providing for a range of constitutive expression. Thus,some are weak constitutive promoters, and others are strong constitutivepromoters. Generally, by “weak promoter” is intended a promoter thatdrives expression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts. Conversely, a “strongpromoter” drives expression of a coding sequence at a “high level,” orabout 1/10 transcripts to about 1/100 transcripts to about 1/1,000transcripts.

One of skill would recognize that modifications could be made to aprotein of the present invention without diminishing its biologicalactivity. Some modifications may be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionsites or termination codons or purification sequences.

Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and thelambda derived P L promoter and N-gene ribosome binding site (Shimatake,et al., (1981) Nature 292:128). The inclusion of selection markers inDNA vectors transfected in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracycline,or chloramphenicol.

The vector is selected to allow introduction of the gene of interestinto the appropriate host cell. Bacterial vectors are typically ofplasmid or phage origin. Appropriate bacterial cells are infected withphage vector particles or transfected with naked phage vector DNA. If aplasmid vector is used, the bacterial cells are transfected with theplasmid vector DNA. Expression systems for expressing a protein of thepresent invention are available using Bacillus sp. and Salmonella(Palva, et al., (1983) Gene 22:229-35; Mosbach, et al., (1983) Nature302:543-5). The pGEX-4T-1 plasmid vector from Pharmacia is the preferredE. coli expression vector for the present invention.

Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, the present invention can be expressedin these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous proteins in yeast is well known. Sherman, etal., (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory isa well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeasts for productionof eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates or the pellets. The monitoring of thepurification process can be accomplished by using Western blottechniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin. Mammaliancell systems often will be in the form of monolayers of cells althoughmammalian cell suspensions may also be used. A number of suitable hostcell lines capable of expressing intact proteins have been developed inthe art, and include the HEK293, BHK21, and CHO cell lines. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter (e.g., the CMV promoter, a HSVtk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer(Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites (e.g., an SV40 large T Ag poly A addition site),and transcriptional terminator sequences. Other animal cells useful forproduction of proteins of the present invention are available, forinstance, from the American Type Culture Collection Catalogue of CellLines and Hybridomas (7^(th) ed., 1992).

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth, andDrosophila cell lines such as a Schneider cell line (see, e.g.,Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-65).

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Otheruseful terminators for practicing this invention include, but are notlimited to, pinII (See An et al. (1989) Plant Cell 1(1):115-122), glb1(See Genbank Accession #L22345), gz (See gzw64a terminator, GenbankAccession #S78780), and the nos terminator from Agrobacterium.

Sequences for accurate splicing of the transcript may also be included.An example of a splicing sequence is the VP1 intron from SV40 (Spragueet al., J. Virol. 45:773-81 (1983)). Additionally, gene sequences tocontrol replication in the host cell may be incorporated into the vectorsuch as those found in bovine papilloma virus type-vectors(Saveria-Campo, “Bovine Papilloma Virus DNA a Eukaryotic CloningVector,” in DNA Cloning: A Practical Approach, vol. II, Glover, ed., IRLPress, Arlington, Va., pp. 213-38 (1985)).

In addition, the NT gene placed in the appropriate plant expressionvector can be used to transform plant cells. The polypeptide can then beisolated from plant callus or the transformed cells can be used toregenerate transgenic plants. Such transgenic plants can be harvested,and the appropriate tissues (seed or leaves, for example) can besubjected to large scale protein extraction and purification techniques.

Plant Transformation Methods

Numerous methods for introducing foreign genes into plants are known andcan be used to insert an NT polynucleotide into a plant host, includingbiological and physical plant transformation protocols. See, e.g., Mikiet al., “Procedure for Introducing Foreign DNA into Plants,” in Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson, eds.,CRC Press, Inc., Boca Raton, pp. 67-88 (1993). The methods chosen varywith the host plant, and include chemical transfection methods such ascalcium phosphate, microorganism-mediated gene transfer such asAgrobacterium (Horsch et al., Science 227:1229-31 (1985)),electroporation, micro-injection, and biolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, e.g., Gruber et al., “Vectors for Plant Transformation,”in Methods in Plant Molecular Biology and Biotechnology, supra, pp.89-119.

The isolated polynucleotides or polypeptides may be introduced into theplant by one or more techniques typically used for direct delivery intocells. Such protocols may vary depending on the type of organism, cell,plant or plant cell, i.e. monocot or dicot, targeted for genemodification. Suitable methods of transforming plant cells includemicroinjection (Crossway, et al., (1986) Biotechniques 4:320-334; andU.S. Pat. No. 6,300,543), electroporation (Riggs, et al., (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606, direct gene transfer (Paszkowski etal., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford, et al., U.S. Pat. No. 4,945,050; WO91/10725; and McCabe, et al., (1988) Biotechnology 6:923-926). Also see,Tomes, et al., “Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment”. pp. 197-213 in Plant Cell, Tissue andOrgan Culture, Fundamental Methods. eds. O. L. Gamborg & G. C. Phillips.Springer-Verlag Berlin Heidelberg New York, 1995; U.S. Pat. No.5,736,369 (meristem); Weissinger, et al., (1988) Ann. Rev. Genet.22:421-477; Sanford, et al., (1987) Particulate Science and Technology5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674(soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein,et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein,et al., (1988) Biotechnology 6:559-563 (maize); WO 91/10725 (maize);Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al.,(1990) Biotechnology 8:833-839; and Gordon-Kamm, et al., (1990) PlantCell 2:603-618 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature(London) 311:763-764; Bytebierm, et al., (1987) Proc. Natl. Acad. Sci.USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) In The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman, et al., pp. 197-209.Longman, N.Y. (pollen); Kaeppler, et al., (1990) Plant Cell Reports9:415-418; and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); U.S. Pat. No. 5,693,512 (sonication);D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li,et al., (1993) Plant Cell Reports 12:250-255; and Christou and Ford,(1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) NatureBiotech. 14:745-750; Agrobacterium mediated maize transformation (U.S.Pat. No. 5,981,840); silicon carbide whisker methods (Frame, et al.,(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)Physiologia Plantarum 93:19-24); sonication methods (Bao, et al., (1997)Ultrasound in Medicine & Biology 23:953-959; Finer and Finer, (2000)Lett Appl Microbiol. 30:406-10; Amoah, et al., (2001) J Exp Bot52:1135-42); polyethylene glycol methods (Krens, et al., (1982) Nature296:72-77); protoplasts of monocot and dicot cells can be transformedusing electroporation (Fromm, et al., (1985) Proc. Natl. Acad. Sci. USA82:5824-5828) and microinjection (Crossway, et al., (1986) Mol. Gen.Genet. 202:179-185); all of which are herein incorporated by reference.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria, which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of plants. See, e.g., Kado,(1991) Crit. Rev. Plant Sci. 10:1. Descriptions of the Agrobacteriumvector systems and methods for Agrobacterium-mediated gene transfer areprovided in Gruber, et al., supra; Miki, et al., supra; and Moloney, etal., (1989) Plant Cell Reports 8:238.

Similarly, the gene can be inserted into the T-DNA region of a Ti or Riplasmid derived from A. tumefaciens or A. rhizogenes, respectively.Thus, expression cassettes can be constructed as above, using theseplasmids. Many control sequences are known which when coupled to aheterologous coding sequence and transformed into a host organism showfidelity in gene expression with respect to tissue/organ specificity ofthe original coding sequence. See, e.g., Benfey and Chua, (1989) Science244:174-81. Particularly suitable control sequences for use in theseplasmids are promoters for constitutive leaf-specific expression of thegene in the various target plants. Other useful control sequencesinclude a promoter and terminator from the nopaline synthase gene (NOS).The NOS promoter and terminator are present in the plasmid pARC2,available from the American Type Culture Collection and designated ATCC67238. If such a system is used, the virulence (vir) gene from eitherthe Ti or Ri plasmid must also be present, either along with the T-DNAportion, or via a binary system where the vir gene is present on aseparate vector. Such systems, vectors for use therein, and methods oftransforming plant cells are described in U.S. Pat. No. 4,658,082; U.S.patent application Ser. No. 913,914, filed Oct. 1, 1986, as referencedin U.S. Pat. No. 5,262,306, issued Nov. 16, 1993; and Simpson, et al.,(1986) Plant Mol. Biol. 6:403-15 (also referenced in the '306 patent);all incorporated by reference in their entirety.

Once constructed, these plasmids can be placed into A. rhizogenes or A.tumefaciens and these vectors used to transform cells of plant species,which are ordinarily susceptible to Fusarium or Alternaria infection.Several other transgenic plants are also contemplated by the presentinvention including but not limited to soybean, corn, sorghum, alfalfa,rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton,melon and pepper. The selection of either A. tumefaciens or A.rhizogenes will depend on the plant being transformed thereby. Ingeneral A. tumefaciens is the preferred organism for transformation.Most dicotyledonous plants, some gymnosperms, and a few monocotyledonousplants (e.g., certain members of the Liliales and Arales) aresusceptible to infection with A. tumefaciens. A. rhizogenes also has awide host range, embracing most dicots and some gymnosperms, whichincludes members of the Leguminosae, Compositae, and Chenopodiaceae.Monocot plants can now be transformed with some success. European PatentApplication No. 604 662 A1 discloses a method for transforming monocotsusing Agrobacterium. European Application No. 672 752 A1 discloses amethod for transforming monocots with Agrobacterium using the scutellumof immature embryos. Ishida, et al., discuss a method for transformingmaize by exposing immature embryos to A. tumefaciens (NatureBiotechnology 14:745-50 (1996)).

Once transformed, these cells can be used to regenerate transgenicplants. For example, whole plants can be infected with these vectors bywounding the plant and then introducing the vector into the wound site.Any part of the plant can be wounded, including leaves, stems and roots.Alternatively, plant tissue, in the form of an explant, such ascotyledonary tissue or leaf disks, can be inoculated with these vectors,and cultured under conditions, which promote plant regeneration. Rootsor shoots transformed by inoculation of plant tissue with A. rhizogenesor A. tumefaciens, containing the gene coding for the fumonisindegradation enzyme, can be used as a source of plant tissue toregenerate fumonisin-resistant transgenic plants, either via somaticembryogenesis or organogenesis. Examples of such methods forregenerating plant tissue are disclosed in Shahin, (1985) Theor. Appl.Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et al., supra; andU.S. patent application Ser. Nos. 913,913 and 913,914, both filed Oct.1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993,the entire disclosures therein incorporated herein by reference.

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice (Hiei, et al.,(1994) The Plant Journal 6:271-82). Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes (Sanford, etal., (1987) Part. Sci. Technol. 5:27; Sanford, (1988) Trends Biotech6:299; Sanford, (1990) Physiol. Plant 79:206; and Klein, et al., (1992)Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang, et al., (1991) BioTechnology 9:996.Alternatively, liposome or spheroplast fusions have been used tointroduce expression vectors into plants. See, e.g., Deshayes, et al.,(1985) EMBO J. 4:2731; and Christou, et al., (1987) Proc. Natl. Acad.Sci. USA 84:3962. Direct uptake of DNA into protoplasts using CaCl₂precipitation, polyvinyl alcohol, or poly-L-ornithine has also beenreported. See, e.g., Hain, et al., (1985) Mol. Gen. Genet. 199:161; andDraper, et al., (1982) Plant Cell Physiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, e.g., Donn, et al., (1990) Abstracts of the VIIth Int'l.Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53;D'Halluin, et al., (1992) Plant Cell 4:1495-505; and Spencer, et al.,(1994) Plant Mol. Biol. 24:51-61.

Increasing the Activity and/or Level of a NT Polypeptide

Methods are provided to increase the activity and/or level of the NTpolypeptide of the invention. An increase in the level and/or activityof the NT polypeptide of the invention can be achieved by providing tothe plant a NT polypeptide. The NT polypeptide can be provided byintroducing the amino acid sequence encoding the NT polypeptide into theplant, introducing into the plant a nucleotide sequence encoding a NTpolypeptide or alternatively by modifying a genomic locus to insert to apolynucleotide encoding the NT polypeptide of the invention.

As discussed elsewhere herein, many methods are known the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, introducing into theplant (transiently or stably) a polynucleotide construct encoding apolypeptide having enhanced nitrogen utilization activity. It is alsorecognized that the methods of the invention may employ a polynucleotidethat is not capable of directing, in the transformed plant, theexpression of a protein or an RNA. Thus, the level and/or activity of aNT polypeptide may be increased by altering the gene encoding the NTpolypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350;Zarling, et al., PCT/US93/03868. Thus, the level and/or activity of a NTpolypeptide may be increased by altering the genome to encode a NTvariant polypeptide.

Reducing the Activity and/or Level of a NT Polypeptide

Methods are provided o reduce or eliminate the activity of an endogenousNT in plant cells, involving the use of NT polynucleotide or polypeptidevariants in conjunction with, but not limited to, transgenic expression,antisense suppression, co-suppression, RNA interference, gene activationor suppression using transcription factors and/or repressors,mutagenesis including transposon tagging, directed and site-specificmutagenesis, chromosome engineering (see Nobrega et. al., Nature431:988-993(04)), homologous recombination, and TILLING.

In accordance with the present invention, the expression of NTpolypeptide is inhibited if the protein level of the NT polypeptide isless than 70% of the protein level of the same NT polypeptide in a plantthat has not been genetically modified or mutagenized to inhibit theexpression of that NT polypeptide. In particular embodiments of theinvention, the protein level of the NT polypeptide in a modified plantaccording to the invention is less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, less than 5%, or lessthan 2% of the protein level of the same NT polypeptide in a plant thatis not a mutant or that has not been genetically modified to inhibit theexpression of that NT polypeptide. The expression level of the NTpolypeptide may be measured directly, for example, by assaying for thelevel of NT polypeptide expressed in the plant cell or plant, orindirectly, for example, by measuring the nitrogen uptake activity ofthe NT polypeptide in the plant cell or plant, or by measuring thephenotypic changes in the plant. Techniques and methods for performingsuch assays are described elsewhere herein and are familiar to oneskilled in the art.

Modulating Root Development

Methods for modulating root development in a plant are provided. By“modulating root development” is intended any alteration in thedevelopment of the plant root when compared to a control plant. Suchalterations in root development include, but are not limited to,alterations in the growth rate of the primary root, the fresh rootweight, the extent of lateral and adventitious root formation, thevasculature system, meristem development, or radial expansion.

Methods for modulating root development in a plant are provided. Themethods comprise modulating the level and/or activity of the NTpolypeptide in the plant. In one method, a NT sequence of the inventionis provided to the plant. In another method, the NT nucleotide sequenceis provided by introducing into the plant a polynucleotide comprising aNT nucleotide sequence of the invention, expressing the NT sequence, andthereby modifying root development. In still other methods, the NTnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant.

In other methods, root development is modulated by altering the level oractivity of the NT polypeptide in the plant. A change in NT activity canresult in at least one or more of the following alterations to rootdevelopment, including, but not limited to, alterations in root biomassand length.

As used herein, “root growth” encompasses all aspects of growth of thedifferent parts that make up the root system at different stages of itsdevelopment in both monocotyledonous and dicotyledonous plants. It is tobe understood that enhanced root growth can result from enhanced growthof one or more of its parts including the primary root, lateral roots,adventitious roots, etc.

Methods of measuring such developmental alterations in the root systemare known in the art. See, for example, U.S. Application No.2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both ofwhich are herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate root development in the plant. Exemplary promotersfor this embodiment include constitutive promoters and root-preferredpromoters. Exemplary root-preferred promoters have been disclosedelsewhere herein.

Furthermore, higher root biomass production due to NT activity has adirect effect on the yield and an indirect effect of production ofcompounds produced by root cells or transgenic root cells or cellcultures of said transgenic root cells. One example of an interestingcompound produced in root cultures is shikonin, the yield of which canbe advantageously enhanced by said methods.

Accordingly, the present invention further provides plants havingmodulated root development when compared to the root development of acontrol plant. In some embodiments, the plant of the invention has anincreased level/activity of the NT polypeptide of the invention and hasenhanced root growth and/or root biomass. In other embodiments, suchplants have stably incorporated into their genome a nucleic acidmolecule comprising a NT nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

Modulating Shoot and Leaf Development

Methods are also provided for modulating shoot and leaf development in aplant. By “modulating shoot and/or leaf development” is intended anyalteration in the development of the plant shoot and/or leaf. Suchalterations in shoot and/or leaf development include, but are notlimited to, alterations in shoot meristem development, in leaf number,leaf size, leaf and stem vasculature, internode length, and leafsenescence. As used herein, “leaf development” and “shoot development”encompasses all aspects of growth of the different parts that make upthe leaf system and the shoot system, respectively, at different stagesof their development, both in monocotyledonous and dicotyledonousplants. Methods for measuring such developmental alterations in theshoot and leaf system are known in the art. See, for example, Werner, etal., (2001) PNAS 98:10487-10492 and U.S. Application No. 2003/0074698,each of which is herein incorporated by reference.

The method for modulating shoot and/or leaf development in a plantcomprises modulating the activity and/or level of a NT polypeptide ofthe invention. In one embodiment, a NT sequence of the invention isprovided. In other embodiments, the NT nucleotide sequence can beprovided by introducing into the plant a polynucleotide comprising a NTnucleotide sequence of the invention, expressing the NT sequence, andthereby modifying shoot and/or leaf development. In other embodiments,the NT nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant.

In specific embodiments, shoot or leaf development is modulated byaltering the level and/or activity of the NT polypeptide in the plant. Achange in NT activity can result in at least one or more of thefollowing alterations in shoot and/or leaf development, including, butnot limited to, changes in leaf number, altered leaf surface, alteredvasculature, internodes and plant growth, and alterations in leafsenescence, when compared to a control plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate shoot and leaf development of the plant. Exemplarypromoters for this embodiment include constitutive promoters,shoot-preferred promoters, shoot meristem-preferred promoters, andleaf-preferred promoters. Exemplary promoters have been disclosedelsewhere herein.

Increasing NT activity and/or level in a plant results in alteredinternodes and growth. Thus, the methods of the invention find use inproducing modified plants. In addition, as discussed above, NT activityin the plant modulates both root and shoot growth. Thus, the presentinvention further provides methods for altering the root/shoot ratio.Shoot or leaf development can further be modulated by altering the leveland/or activity of the NT polypeptide in the plant.

Accordingly, the present invention further provides plants havingmodulated shoot and/or leaf development when compared to a controlplant. In some embodiments, the plant of the invention has an increasedlevel/activity of the NT polypeptide of the invention. In otherembodiments, the plant of the invention has a decreased level/activityof the NT polypeptide of the invention.

Modulating Reproductive Tissue Development

Methods for modulating reproductive tissue development are provided. Inone embodiment, methods are provided to modulate floral development in aplant. By “modulating floral development” is intended any alteration ina structure of a plant's reproductive tissue as compared to a controlplant in which the activity or level of the NT polypeptide has not beenmodulated. “Modulating floral development” further includes anyalteration in the timing of the development of a plant's reproductivetissue (i.e., a delayed or a accelerated timing of floral development)when compared to a control plant in which the activity or level of theNT polypeptide has not been modulated. Macroscopic alterations mayinclude changes in size, shape, number, or location of reproductiveorgans, the developmental time period that these structures form, or theability to maintain or proceed through the flowering process in times ofenvironmental stress. Microscopic alterations may include changes to thetypes or shapes of cells that make up the reproductive organs.

The method for modulating floral development in a plant comprisesmodulating NT activity in a plant. In one method, a NT sequence of theinvention is provided. A NT nucleotide sequence can be provided byintroducing into the plant a polynucleotide comprising a NT nucleotidesequence of the invention, expressing the NT sequence, and therebymodifying floral development. In other embodiments, the NT nucleotideconstruct introduced into the plant is stably incorporated into thegenome of the plant.

In specific methods, floral development is modulated by increasing thelevel or activity of the NT polypeptide in the plant. A change in NTactivity can result in at least one or more of the following alterationsin floral development, including, but not limited to, altered flowering,changed number of flowers, modified male sterility, and altered seedset, when compared to a control plant. Inducing delayed flowering orinhibiting flowering can be used to enhance yield in forage crops suchas alfalfa. Methods for measuring such developmental alterations infloral development are known in the art. See, for example, Mouradov, etal., (2002) The Plant Cell S111-S130, herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate floral development of the plant. Exemplary promotersfor this embodiment include constitutive promoters, inducible promoters,shoot-preferred promoters, and inflorescence-preferred promoters.

In other methods, floral development is modulated by altering the leveland/or activity of the NT sequence of the invention. Such methods cancomprise introducing a NT nucleotide sequence into the plant andchanging the activity of the NT polypeptide. In other methods, the NTnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant. Altering expression of the NT sequence ofthe invention can modulate floral development during periods of stress.Such methods are described elsewhere herein. Accordingly, the presentinvention further provides plants having modulated floral developmentwhen compared to the floral development of a control plant. Compositionsinclude plants having a altered level/activity of the NT polypeptide ofthe invention and having an altered floral development. Compositionsalso include plants having a modified level/activity of the NTpolypeptide of the invention wherein the plant maintains or proceedsthrough the flowering process in times of stress.

Methods are also provided for the use of the NT sequences of theinvention to increase seed size and/or weight. The method comprisesincreasing the activity of the NT sequences in a plant or plant part,such as the seed. An increase in seed size and/or weight comprises anincreased size or weight of the seed and/or an increase in the size orweight of one or more seed part including, for example, the embryo,endosperm, seed coat, aleurone, or cotyledon.

As discussed above, one of skill will recognize the appropriate promoterto use to increase seed size and/or seed weight. Exemplary promoters ofthis embodiment include constitutive promoters, inducible promoters,seed-preferred promoters, embryo-preferred promoters, andendosperm-preferred promoters.

The method for altering seed size and/or seed weight in a plantcomprises increasing NT activity in the plant. In one embodiment, the NTnucleotide sequence can be provided by introducing into the plant apolynucleotide comprising a NT nucleotide sequence of the invention,expressing the NT sequence, and thereby decreasing seed weight and/orsize. In other embodiments, the NT nucleotide construct introduced intothe plant is stably incorporated into the genome of the plant.

It is further recognized that increasing seed size and/or weight canalso be accompanied by an increase in the speed of growth of seedlingsor an increase in early vigor. As used herein, the term “early vigor”refers to the ability of a plant to grow rapidly during earlydevelopment, and relates to the successful establishment, aftergermination, of a well-developed root system and a well-developedphotosynthetic apparatus. In addition, an increase in seed size and/orweight can also result in an increase in plant yield when compared to acontrol.

Accordingly, the present invention further provides plants having anincreased seed weight and/or seed size when compared to a control plant.In other embodiments, plants having an increased vigor and plant yieldare also provided. In some embodiments, the plant of the invention has amodified level/activity of the NT polypeptide of the invention and hasan increased seed weight and/or seed size. In other embodiments, suchplants have stably incorporated into their genome a nucleic acidmolecule comprising a NT nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

Method of Use for NT Polynucleotide, Expression Cassettes, andAdditional Polynucleotides

In certain embodiments the nucleic acid sequences of the presentinvention can be stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredphenotype. The combinations generated may include multiple copies of anyone of the polynucleotides of interest. For example, a polynucleotide ofthe present invention may be stacked with any other polynucleotide(s) ofthe present invention. The polynucleotides of the present invention maybe stacked with any gene or combination of genes to produce plants witha variety of desired trait combinations, including but not limited totraits desirable for animal feed such as high oil genes (e.g., U.S. Pat.No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat.Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine(Williamson, et al., (1987) Eur. J. Biochem. 165:99-106; and WO98/20122); and high methionine proteins (Pedersen, et al., (1986) J.Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359; andMusumura, et al., (1989) Plant Mol. Biol. 12:123)); increaseddigestibility (e.g., modified storage proteins (U.S. application Ser.No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. applicationSer. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which areherein incorporated by reference. The polynucleotides of the presentinvention can also be stacked with traits desirable for insect, diseaseor herbicide resistance (e.g., Bacillus thuringiensis toxic proteins(U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881;Geiser, et al., (1986) Gene 48:109); lectins (Van Damme, et al., (1994)Plant Mol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No.5,792,931); avirulence and disease resistance genes (Jones, et al.,(1994) Science 266:789; Martin, et al., (1993) Science 262:1432;Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase (ALS)mutants that lead to herbicide resistance such as the S4 and/or Hramutations; inhibitors of glutamine synthase such as phosphinothricin orbasta (e.g., bar gene); and glyphosate resistance (EPSPS gene)); andtraits desirable for processing or process products such as high oil(e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE) and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosuresof which are herein incorporated by reference. One could also combinethe polynucleotides of the present invention with polynucleotidesaffecting agronomic traits such as male sterility (e.g., see U.S. Pat.No. 5,583,210), stalk strength, flowering time, or transformationtechnology traits such as cell cycle regulation or gene targeting (e.g.,WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which areherein incorporated by reference.

In one embodiment, the polynucleotides of the present invention may bestacked with one or more polynucleotides that improve nitrate uptake ornitrogen assimilation or both. Such polynucleotides include genes or thecoding regions of, including but not limited to, nitrate transporters,nitrate reductases and/or root genes. Exemplary nitrate transportersinclude without limitation YNT1, optimized or partially optimized YNT1and variants thereof. Exemplary nitrate reductases include withoutlimitation YNR1 and PPNR A551G which are described elsewhere herein.Exemplary root genes include without limitation maize genomic clone ofcytokinin oxidase/dehydrogenase gene (ZM-CKXg).

In one embodiment, sequences of interest improve plant growth and/orcrop yields. For example, sequences of interest include agronomicallyimportant genes that result in improved primary or lateral root systems.Such genes include, but are not limited to, nutrient/water transportersand growth induces. Examples of such genes, include but are not limitedto, maize plasma membrane H⁺-ATPase (MHA2) (Frias, et al., (1996) PlantCell 8:1533-44); AKT1, a component of the potassium uptake apparatus inArabidopsis, (Spalding, et al., (1999) J Gen Physiol 113:909-18); RMLgenes which activate cell division cycle in the root apical cells(Cheng, et al., (1995) Plant Physiol 108:881); maize glutaminesynthetase genes (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46) andhemoglobin (Duff, et al., (1997) J. Biol. Chem. 27:16749-16752,Arredondo-Peter, et al., (1997) Plant Physiol. 115:1259-1266;Arredondo-Peter, et al., (1997) Plant Physiol 114:493-500 and referencessited therein). The sequence of interest may also be useful inexpressing antisense nucleotide sequences of genes that that negativelyaffects root development.

Additional, agronomically important traits such as oil, starch, andprotein content can be genetically altered in addition to usingtraditional breeding methods. Modifications include increasing contentof oleic acid, saturated and unsaturated oils, increasing levels oflysine and sulfur, providing essential amino acids, and alsomodification of starch. Hordothionin protein modifications are describedin U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, hereinincorporated by reference. Another example is lysine and/or sulfur richseed protein encoded by the soybean 2S albumin described in U.S. Pat.No. 5,850,016, and the chymotrypsin inhibitor from barley, described inWilliamson, et al., (1987) Eur. J. Biochem. 165:99-106, the disclosuresof which are herein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley, et al., (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502; herein incorporated by reference); corn (Pedersen, et al.,(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359;both of which are herein incorporated by reference); and rice (Musumura,et al., (1989) Plant Mol. Biol. 12:123, herein incorporated byreference). Other agronomically important genes encode latex, Floury 2,growth factors, seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser, et al., (1986) Gene 48:109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones, et al., (1994) Science 266:789;Martin, et al., (1993) Science 262:1432; and Mindrinos, et al., (1994)Cell 78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta, the nptII gene encodes resistance to theantibiotics kanamycin and geneticin, and the ALS-gene mutants encoderesistance to the herbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see,Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHA5).

Exogenous products include plant enzymes and products as well as thosefrom other sources including procaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

Genes or coding sequences that are stacked with the NTs described hereinmay be driven by any suitable promoter that expresses the polynucleotidein the desired temporal, spatial pattern and level within the plant orplant cell. Modification of gene expression in nitrate assimilationpathway, e.g. nitrate reductase and/or root genes along with NT mayimprove NUE more efficiently. Exemplary stacking constructs include butare not limited to the following set forth below.

TABLE 2 Exemplary stacking constructs. Promoter for Additional Promoterfor Additional Coding expression Coding expression Coding Constructsequence or of stacked sequence or of stacked sequence or identifierpromoter gene of NT gene gene gene gene PHP38942 ZM-RM2¹ YNT1 ZM-PEPCPPNR ADHI Intron A551G PHP38943 BSV YNT1 ZM-PEPC PPNR (TR):ADHI A551GIntron PHP38945 ZM- YNT1 BSV ZM-CKXg ZM-PEPC PPNR RM2:ADHI (TR):ADHIA551G Intron Intron PHP new ZM-RM2 YNT1 ZM-PEPC PPNR ADHI Intron A551GS561D MO ¹Maize root metallothionein promoter = ZM-RM2; maize NAS2promoter, banana streak virus promoter truncated version promoter = BSV(TR), maize phosphoenolpyruvate carboxylase promoter = ZM-PEPC; maizeubiquitin promoter = UBI (maize ubiquitin promoter (Christensen et al.,Plant Mol. Biol. 12: 619-632 (1989) and Christensen et al., Plant Mol.Biol. 18: 675-689 (1992)); ADHI Intron = Intron of alcohol dehydrogenase1 gene; ZM-RM2 promoter may be used in combination with an ADHI Intron;maize genomic clone of cytokinin oxidase/dehydrogenase gene = ZM-CKXg;PPNR A551G, also referred to as A7G PPNR = wild type red algae nitratereductase (Porphyra perforata) wherein the seventh alanine in theputative allergen site of wild type PPNR has been substituted with aglycine amino acid at that position. PPNR A551G S561D MO = maize codonoptimized wild type red algae nitrate reductase (Porphyra perforata)wherein the seventh alanine in the putative allergen site of wild typePPNR has been substituted with a glycine amino acid at position 551 andthe serine residue at position 561 has been substituted with asparticacid amino acid to knock out the putative phosphorylation site. SeeFIGS. 4 and 5. Wild type PPNR contains a putative allergen peptide with8 aa residues, Val followed by 7 Alas, Val Ala1 Ala2 Ala3 Ala4 Ala5 Ala6Ala7. See FIG. 5.

The PPNR A551G or A7G PPNR and PPNR A551G S561D MO have “NR activity”which refers to an activity exerted by a Nitrate Reductase protein,polypeptide or portion thereof as determined in vivo, or in vitro,according to standard techniques. In one aspect, NR activity is thereduction of nitrate to nitrite. In one aspect, NR activity includes butis not limited to increased nitrate reduction rate and/or specificityfor nitrate, for example, decreased K_(m) for nitrate and NADH,increased velocity (V_(max)) for nitrate reduction and the like ascompared to NR activity of an endogenous NR of a crop plant of interest.In another aspect, NR activity includes but is not limited to increasingNUE and/or plant productivity/yield as compared to a control plant. NUEmay be inferred from amount and/or rate of nitrogen uptake from the soilor medium. In another aspect, NR activity includes but is not limited tomaintaining NR activity, for example, as compared to a wild type NR,while inactivating post-translational regulation by knocking out theputative serine residue, e.g. Ser 561 of PPNR when consumed as comparedto a control plant, e.g. expressing a wild type NR. Methods andtechniques for testing for NR activity will be known to one skilled inthe art and are also described in U.S. patent application Ser. No.12/138,477, filed Jun. 13, 2008, herein incorporated by reference in itsentirety.

The polynucleotides of the present invention can be used in thetransformation of any plant species, including, but not limited to,monocots and dicots. Examples of plant species of interest include, butare not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean andsugarcane plants are optimal, and in yet other embodiments corn plantsare optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpeachickpea, etc.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. The disclosure of each reference set forthherein is incorporated herein by reference in its entirety.

EXAMPLES

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

Example 1 Cloning of Yeast Nitrate Transporter (YNT1) Coding Sequencefrom Pichia angusta and Test its Functionality in Pichia pastoris

PCR was used to obtain the coding sequence of YNT1 gene from Pichiaangusta genomic DNA based on the published data (Perez M D et al.,Biochem. J., (1997) 15:397-403) with BamH I and EcoRI restriction sitesat the respective 5′- and 3′ ends. The fragment was cloned intopCR-Blunt TOPO vector for sequencing. The functionality of YNT1 wasverified using Pichia pastoris system developed at Pioneer Hi-Bred Int'l(U.S. patent application Ser. No. 12/136,173). The clone with correctsequence was used to make yeast expression vector pGAPZA-YNT1 via BamHIand EcoRI sites. Pichia pastoris strain KM71 (Invitrogen) carryingp3.5GAP-YNR1 (yeast nitrate reductase driven by pGAP promoter integratedinto His4 locus) was transformed by pGAPZA-YNT1 via integration into thepGAP promoter region to generate KM71 strain carrying both YNT1 and YNR1gene expression cassettes. Functional transformants were identified bynitrate uptake assay in vivo (U.S. patent application Ser. No.12/136,173).

Example 2 YNT1 Coding Sequence Modification for Maize Expression

To enhance the expression potential in maize, the codon of YNT1 codingsequence was optimized for maize expression. The rare codons wereeliminated. The GC composition was targeted to 60% and distributedrelative flat over the length of the ORF. At same time, several unwantedfeatures including cryptic intron donor or acceptor sites, RNAinstability sites, long homogenous base stretches, and undesiredrestriction enzymes were removed. The sequence of maize codon optimizedYNT1 (YNT1 MO) is shown in SEQ ID NO:3. The maize expression constructsof YNT1 MO are driven by root-preferred promoter, e.g. ZM-RM2 promoterand BSV (TR) promoter was prepared by standard cloning techniques.

Example 3 Preparation of a Plant Expression Vector

Two expression vectors of YNT1 gene driven by maize UBI promoter(PHP26091) or maize ZM-RM2 promoter (PHP27279) were made by standardcloning techniques for maize GapexGS3 transformation.

More constructs of YNT1 driven by different promoters were made forelite maize line transformation. They are PHP32095 (ZM-RM2:YNT1),PHP32100 (ZM-RM2:ADHI Intron:YNT1), PHP38318 (BSV (TR):ADHIIntron:YNT1), PHP38506 (ZM-NAS2:YNT1), new PHP (BAV (FL): YNT1).

Several stacking constructs including nitrate transporter gene, nitratereductase gene and/or root gene to improve nitrate uptake andassimilation were made for elite line transformation. They are PHP32372(ZM-RM2:ADHI Intron:YNT1//UBI:YNR1), PHP32267 (ZM-RM2:YNT1//UBI:YNR1),PHP38942 (ZM-PEPC:PPNR A551G//ZM-RM2:ADHI Intron:YNT1), PHP38943(ZM-PEPC:PPNR A551G//BSV (TR):YNT1), PHP38945 (BSV(TR):ZM-CKXg//ZM-RM2:ADHI Intron:YNT1//ZM-PEPC:PPNR A551G).

Example 4 Agrobacterium Mediated Transformation of Maize with NTs(Prophetic)

Agrobacterium-mediated transformation of maize is performed essentiallyas described by Zhao et al., in Meth. Mol. Biol. 318:315-323 (2006) (seealso Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No.5,981,840 issued Nov. 9, 1999, incorporated herein by reference). Thetransformation process involves bacterium inoculation, co-cultivation,resting, selection and plant regeneration.

1. Immature Embryo Preparation

Immature embryos are dissected from caryopses and placed in a 2 mLmicrotube containing 2 mL PHI-A medium.

2. Agrobacterium Infection and Co-Cultivation of Embryos

2.1 Infection Step

PHI-A medium is removed with 1 mL micropipettor and 1 mL Agrobacteriumsuspension is added. Tube is gently inverted to mix. The mixture isincubated for 5 min at room temperature.

2.2 Co-Culture Step

The Agrobacterium suspension is removed from the infection step with a 1mL micropipettor. Using a sterile spatula the embryos are scraped fromthe tube and transferred to a plate of PHI-B medium in a 100×15 mm Petridish. The embryos are oriented with the embryonic axis down on thesurface of the medium. Plates with the embryos are cultured at 20° C.,in darkness, for 3 days. L-Cysteine can be used in the co-cultivationphase. With the standard binary vector, the co-cultivation mediumsupplied with 100-400 mg/L L-cysteine is critical for recovering stabletransgenic events.

3. Selection of Putative Transgenic Events

To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos aretransferred, maintaining orientation and the dishes are sealed withParafilm. The plates are incubated in darkness at 28° C. Activelygrowing putative events, as pale yellow embryonic tissue are expected tobe visible in 6-8 weeks. Embryos that produce no events may be brown andnecrotic, and little friable tissue growth is evident. Putativetransgenic embryonic tissue is subcultured to fresh PHI-D plates at 2-3week intervals, depending on growth rate. The events are recorded.

4. Regeneration of T0 plants

Embryonic tissue propagated on PHI-D medium is subcultured to PHI-Emedium (somatic embryo maturation medium); in 100×25 mm Petri dishes andincubated at 28° C., in darkness, until somatic embryos mature, forabout 10-18 days. Individual, matured somatic embryos with well-definedscutellum and coleoptile are transferred to PHI-F embryo germinationmedium and incubated at 28° C. in the light (about 80 μE from cool whiteor equivalent fluorescent lamps). In 7-10 days, regenerated plants,about 10 cm tall, are potted in horticultural mix and hardened-off usingstandard horticultural methods.

Media for Plant Transformation

-   -   1. PHI-A: 4 g/L CHU basal salts, 1.0 mL/L 1000× Eriksson's        vitamin mix, 0.5 mg/L thiamin HCL, 1.5 mg/L 2,4-D, 0.69 g/L        L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 μM        acetosyringone, filter-sterilized before using.    -   2. PHI-B: PHI-A without glucose, increased 2,4-D to 2 mg/L,        reduced sucrose to 30 g/L and supplemented with 0.85 mg/L silver        nitrate (filter-sterilized), 3.0 g/L gelrite, 100 μM        acetosyringone (filter-sterilized), 5.8.    -   3. PHI-C: PHI-B without gelrite and acetosyringonee, reduced        2,4-D to 1.5 mg/L and supplemented with 8.0 g/L agar, 0.5 g/L        Ms-morpholino ethane sulfonic acid (MES) buffer, 100 mg/L        carbenicillin (filter-sterilized).    -   4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos        (filter-sterilized).    -   5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL        11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5        mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5        mg/L zeatin (Sigma, cat. no. Z-0164), 1 mg/L indole acetic acid        (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L        bialaphos (filter-sterilized), 100 mg/L carbenicillin        (fileter-sterilized), 8 g/L agar, pH 5.6.    -   6. PHI-F: PHI-E without zeatin, IAA, ABA; sucrose reduced to 40        g/L; replacing agar with 1.5 g/L gelrite; pH 5.6.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Phenotypic analysis of transgenic T0 plants and T1 plants can beperformed.

T1 plants can be analyzed for phenotypic changes. Using image analysisT1 plants can be analyzed for phenotypical changes in plant area,volume, growth rate and color analysis can be taken at multiple timesduring growth of the plants. Alteration in root architecture can beassayed as described herein.

Subsequent analysis of alterations in agronomic characteristics can bedone to determine whether plants containing a NT polynucleotidedescribed herein have an improvement of at least one agronomiccharacteristic, when compared to the control (or reference) plants thatdo not contain the NT polynucleotide. The alterations may also bestudied under various environmental conditions.

Expression constructs containing the NT polynucleotide that result in asignificant alteration in root and/or shoot biomass, improved greencolor, larger ear at anthesis or yield will be considered evidence thatthe NT polynucleotide functions in maize to alter nitrogen useefficiency or nitrate uptake.

Example 5 Transformation of Maize with NTs Using Particle Bombardment(Prophetic)

Maize plants can be transformed to express or overexpress a NTpolynucleotide described herein in order to examine the resultingphenotype.

Expression of the gene in maize can be under control of a constitutivepromoter such as the maize ubiquitin promoter (Christensen et al., PlantMol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol.18:675-689 (1992))

The recombinant DNA construct described above can then be introducedinto maize cells by the following procedure. Immature maize embryos canbe dissected from developing caryopses derived from crosses of theinbred maize lines H99 and LH132. The embryos are isolated ten to elevendays after pollination when they are 1.0 to 1.5 mm long. The embryos arethen placed with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al., Sci. Sin. Peking 18:659-668(1975)). The embryos are kept in the dark at 27° C. Friable embryogeniccallus consisting of undifferentiated masses of cells with somaticproembryoids and embryoids borne on suspensor structures proliferatesfrom the scutellum of these immature embryos. The embryogenic callusisolated from the primary explant can be cultured on N6 medium andsub-cultured on this medium every two to three weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from cauliflower mosaic virus (Odell et al., Nature313:810-812 (1985)) and the 3′ region of the nopaline synthase gene fromthe T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al., Nature 327:70-73 (1987))may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After ten minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the maize tissue with a Biolistic® PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialaphos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additionaltwo weeks the tissue can be transferred to fresh N6 medium containingbialaphos. After six weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialaphos-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).Transgenic T0 plants can be regenerated and their phenotype determinedfollowing HTP procedures. T1 seed can be collected.

T1 plants can be grown and analyzed for phenotypic changes. Thefollowing parameters can be quantified using image analysis: plant area,volume, growth rate and color analysis can be collected and quantified.Expression constructs that result in an alteration of root architectureor any one of the agronomic characteristics listed above compared tosuitable control plants, can be considered evidence that the NTpolynucleotide functions in maize to alter root architecture or plantarchitecture.

Furthermore, a recombinant DNA construct containing a NT polynucleotidedescribed herein can be introduced into an maize line either by directtransformation or introgression from a separately transformed line.

Transgenic plants, either inbred or hybrid, can undergo more vigorousfield-based experiments to study root or plant architecture, yieldenhancement and/or resistance to root lodging under variousenvironmental conditions (e.g. variations in nutrient and wateravailability).

Subsequent yield analysis can also be done to determine whether plantsthat contain the NT polynucleotide have an improvement in yieldperformance, when compared to the control (or reference) plants that donot contain the NT polynucleotide. Plants containing the NTpolynucleotide would improved yield relative to the control plants,preferably 50% less yield loss under adverse environmental conditions orwould have increased yield relative to the control plants under varyingenvironmental conditions.

Example 6 Electroporation of Agrobacterium tumefaciens LBA4404(Prophetic)

Electroporation competent cells (40 μl), such as Agrobacteriumtumefaciens LBA4404 (containing PHP10523), are thawn on ice (20-30 min).PHP10523 contains VIR genes for T-DNA transfer, an Agrobacterium lowcopy number plasmid origin of replication, a tetracycline resistancegene, and a cos site for in vivo DNA biomolecular recombination.Meanwhile the electroporation cuvette is chilled on ice. Theelectroporator settings are adjusted to 2.1 kV.

A DNA aliquot (0.5 μL JT (U.S. Pat. No. 7,087,812) parental DNA at aconcentration of 0.2 μg-1.0 μg in low salt buffer or twice distilledH₂O) is mixed with the thawn Agrobacterium cells while still on ice. Themix is transferred to the bottom of electroporation cuvette and kept atrest on ice for 1-2 min. The cells are electroporated (Eppendorfelectroporator 2510) by pushing “Pulse” button twice (ideally achievinga 4.0 msec pulse). Subsequently 0.5 ml 2×YT medium (or SOCmedium) areadded to cuvette and transferred to a 15 ml Falcon tube. The cells areincubated at 28-30° C., 200-250 rpm for 3 h.

Aliquots of 250 μl are spread onto #30B (YM+50 μg/mL Spectinomycin)plates and incubated 3 days at 28-30° C. To increase the number oftransformants one of two optional steps can be performed:

Option 1: overlay plates with 30 μl of 15 mg/ml Rifampicin. LBA4404 hasa chromosomal resistance gene for Rifampicin. This additional selectioneliminates some contaminating colonies observed when using poorerpreparations of LBA4404 competent cells.

Option 2: Perform two replicates of the electroporation to compensatefor poorer electrocompetent cells.

Identification of Transformants:

Four independent colonies are picked and streaked on AB minimal mediumplus 50 mg/mL Spectinomycin plates (#12S medium) for isolation of singlecolonies. The plated are incubate at 28° C. for 2-3 days.

A single colony for each putative co-integrate is picked and inoculatedwith 4 ml #60A with 50 mg/l Spectinomycin. The mix is incubated for 24 hat 28° C. with shaking Plasmid DNA from 4 ml of culture is isolatedusing Qiagen Miniprep+optional PB wash. The DNA is eluted in 30 μl.Aliquots of 2 μl are used to electroporate 20 μl of DH10b+20 μl of ddH₂Oas per above.

Optionally a 15 μl aliquot can be used to transform 75-100 μl ofInvitrogen™-Library Efficiency DH5α. The cells are spread on LB mediumplus 50 mg/mL Spectinomycin plates (#34T medium) and incubated at 37° C.overnight.

Three to four independent colonies are picked for each putativeco-integrate and inoculated 4 ml of 2×YT (#60A) with 50 μg/mlSpectinomycin. The cells are incubated at 37° C. overnight with shaking.

The plasmid DNA is isolated from 4 ml of culture using QIAprep® Miniprepwith optional PB wash (elute in 50 μl) and 8 μl are used for digestionwith SalI (using JT parent and PHP10523 as controls).

Three more digestions using restriction enzymes BamHI, EcoRI, andHindIII are performed for 4 plasmids that represent 2 putativeco-integrates with correct SalI digestion pattern (using parental DNAand PHP10523 as controls). Electronic gels are recommended forcomparison.

Example 7 Transgenic Plants Evaluation Using Uptake Assay in Arabidopsis

YNT1 gene was cloned into Arabidopsis expression vector (pMAXY5295)under the control of pTUB promoter a root preferred Arabidopsispromoter. Following the standard Agrobacterium transformation, multipleevents were recovered.

Briefly, the construct containing pTUB:YNT1 was transformed intoAgrobacterium tumefaciens strain C58, grown in LB at 25° C. to OD600˜1.0. Cells were then pelleted by centrifugation and resuspended in anequal volume of 5% sucrose/0.05% Silwet L-77 (OSI Specialties, Inc). Atearly bolting, soil grown Arabidopsis thaliana ecotype Col-0 were dippedinto the Agrobacterium suspension. The plants were then allowed to setseed as normal. The resulting T₁ seed were sown on soil, and transgenicseedlings were selected by growing T1 seeds on medium with Kanamycin.The resistant seedlings were transplanted into soil. T2 seeds werecollected from T1 plates resistant to Kanamycin selection. T₂ seed wascollected,

The presence of the transgene was analyzed by RT-PCR and Western-blotanalysis. Using the protocol detailed in U.S. patent application Ser.No. 12/166,473, filed Jul. 3, 2007, a pH-dye based nitrate uptakeseedling assay was performed for those transformant events. Multipleevents (3 out of 8) from pTUB-YNT1-R3FS construct demonstratedstatistically significant enhancement in nitrate uptake when comparedwith nitrate uptake of the wild type YNT construct.

Example 8 Low Nitrate Assay in GS3× Gaspe to Determine Shoot and Ear DryWeight

Transgenic plants contain two or three doses of Gaspe Flint-3 with onedose of GS3 (GS3/(Gaspe-3)2× or GS3/(Gaspe-3)3×) and segregate 1:1 for adominant transgene.

Transgenic GS3× Gaspe T1 seeds were planted in 4 inch pots containingTurface and watered with nutrient solution (Table 3) containing 1 mMnitrate as the sole nitrogen source for 2 weeks.

TABLE 3 Nutrient Solution Nutrient Concentration KNO₃ 1 mM KCl 3 mMMgSO₄ 2 mM CaCl₂ 2 mM KH₂PO₄ 0.5 mM Chelated Iron 8.3 g 100 l⁻¹ MnSO₄0.5 μM ZnSO₄ 0.5 μM H₃BO₄ 1.5 μM CuSO₄ 0.05 μM H₂MoO₄ 0.05 μM

YNT1 and its respective nulls were grown in each block. The nutrientswere replaced 4 times each day to maintain a constant concentration ofnutrients. After emergence plants were sampled to determine which weretransgenic and which were nulls. At anthesis plants were harvested anddried in a 70° C. oven for 72 hr and the shoot and ear dry weightdetermined. Transgene means were calculated and compared to the grandmean, the block mean and their respective null means (see Table 4).Control plants grown in 1 mM KNO₃ medium. Statistical analysis wasperformed to determine if observed differences between treatments aresignificant. Improvements in biomass and ear size at anthesis isindicative of increased nitrogen tolerance.

Example 9 Green House Low Nitrate Assay to Determine Root Dry Weight,Shoot Dry Weight, Root/Shoot Ratio, Total Plant Weight, Total NConcentration and Total Plant N

Transgenic T1/T2 Plants were grown in nutrient medium (Table 3)containing 1 mM nitrate as the sole nitrogen source for 2 weeks. After 2weeks the plants were harvested and root dry weight, shoot dry weight,root/shoot ratio, total plant weight, total N concentration and totalplant N determined. Data was analyzed using a nearest neighbor analysisto estimate the variance. Transgenic means were compared to theexperiment grand mean, to the block mean after the mean in question wasremoved from the block mean estimate, and to the correspondingtransgenic null mean. Table 4 shows the Student's t probabilitycomparing the transgenic means to the corresponding null means.

TABLE 4 YNT1 Gaspe Means. Transgene Grand Block Null Component Mean MeanMean Mean SID 11327791 Ear Dwt 2.1157 0.8974 1.2466 1.3944 (P > t)<0.001 0.001 0.081 Plant Dwt 13.7 11.8 13.7 12.9 (P > t) 0.002 NS NS SID11260358 Ear Dwt 1.4 0.8974 0.7986 0.8473 (P > t) NS 0.097 NS Plant Dwt10.9 11.8 11.1 9.6 (P > t) NS NS NS SID 11350779 Ear Dwt 1.0515 0.89740.7766 0.5159 (P > t) NS NS NS Plant Dwt 11.8 11.8 11 9.1 (P > t) NS NS0.008 SID 11260374 Ear Dwt 1.3 0.8974 0.7986 0.5526 (P > t) NS 0.0730.067 Plant Dwt 10.8 11.8 11.1 9.3 (P > t) NS NS 0.08 SID 11260371 EarDwt 1.325 0.8974 1.2466 0.7414 (P > t) NS NS NS Plant Dwt 12.5 11.8 13.714.9 (P > t) NS 0.067 0.016 SID 11350777 Ear Dwt 0.6158 0.8974 0.77660.9 (P > t) NS NS NS Plant Dwt 12.2 11.8 11 9.4 (P > t) NS 0.052 0.003SID 11327795 Ear Dwt 1.021 0.8974 1.2466 1.5784 (P > t) NS NS NS PlantDwt 16 11.8 13.7 12.3 (P > t) <0.001 0.002 <0.001 SID 11260359 Ear Dwt0.8866 0.8974 0.7766 0.433 (P > t) NS NS NS Plant Dwt 10/5 11.8 11 10.2(P > t) NS NS NS

Any mean with a Student's t probability 0.1 or less is listed in thetable and any values with a Student's t probability greater than 0.1 arelisted as non significant (NS). Student's t probabilities of transgenemeans greater than the null means are designated with an asterisk (*).The data from PHP32100 (Table 5), and PHP32095 (Table 6) are summarizedhere.

TABLE 5 GH LN assay PHP32100 Root Root Sig Shoot Shoot Sig Total TotalSig Event Dwt Dwt Null Level Dwt Dwt Null Level Dwt Dwt Null Level 10.84 0.86 NS 1.81 1.85 NS 2.65 2.71 NS 2 0.91 0.86 0.019* 1.98 1.850.024* 2.89 2.71 0.020* 3 0.94 0.86 0.001* 1.97 1.85 0.039* 2.91 2.710.014* 4 0.97 0.86 <0.001* 2.03 1.85 0.004* 2.99 2.71 <0.001* 5 0.950.86 <0.001* 2.03 1.85 0.004* 2.98 2.71 0.001* 6 0.94 0.86 <0.001* 2.061.85 <0.001* 3 2.71 <0.001* 7 0.93 0.86 0.002* 2.06 1.85 0.001* 2.992.71 <0.001* 8 0.95 0.86 <0.001* 2.11 1.85 <0.001* 3.06 2.71 <0.001* 90.83 0.86 NS 1.87 1.85 NS 2.7 2.71 NS 10  0.91 0.86 0.017* 2.05 1.850.002* 2.96 2.71 0.003* Total N Root/ Root/Shoot Sig mg N/g mg N/g SigTotal N (mg) Sig Event Shoot Null Level Dwt Dwt Null Level (mg) NullLevel 1 0.468 0.4609 NS 19.6160 21.4400 0.03 52.106 55.49 NS 2 0.46040.4609 NS 18.3590 21.4400 0.00 52.956 55.49 NS 3 0.4783 0.4609 0.018*21.3710 21.4400 NS 61.837 55.49 0.010* 4 0.4833 0.4609 0.004* 18.125021.4400 <0.001 53.413 55.49 NS 5 0.4717 0.4609 0.095* 16.0060 21.4400<0.001 47.699 55.49 0.00 6 0.4576 0.4609 NS 20.0340 21.4400 0.08 59.48155.49 0.071* 7 0.4587 0.4609 NS 21.7890 21.4400 NS 65.161 55.49 <0.001*8 0.4575 0.4609 NS 18.8620 21.4400 0.01 57.937 55.49 NS 9 0.4474 0.46090.05 20.0360 21.4400 0.08 52.661 55.49 NS 10  0.4482 0.4609 0.06 19.885021.4400 0.06 58.769 55.49 NS Root Root Sig Shoot Shoot Sig Total TotalSig Event Dwt Dwt Null Level Dwt Dwt Null Level Dwt Dwt Null Level 11.45 1.25 0.010* 3.37 3.16 NS 4.83 4.41 0.052* 2 1.25 1.25 NS 3.2 3.16NS 4.46 4.41 NS 3 1.37 1.25 0.073* 3.42 3.16 0.073* 4.8 4.41 0.065* 41.31 1.25 NS 3.29 3.16 NS 4.6 4.41 NS 5 1.46 1.25 0.007* 3.9 3.16<0.001* 5.36 4.41 <0.001*

TABLE 6 GH LN assay PHP32095 Root/ Total N Root/ Shoot Sig mg N/g mg N/gDwt Total N (mg) Sig Event Shoot Null Level Dwt Null Sig Level (mg) NullLevel 1 0.4243 0.3909 0.011* 18.6530 18.3360 NS 77.9462 79.229 NS 20.3892 0.3909 NS 18.9420 18.3360 NS 81.4487 79.229 NS 3 0.4033 0.3909 NS17.9010 18.3360 NS 85.0412 79.229 0.034* 4 0.3993 0.3909 NS 18.741018.3360 NS 83.2311 79.229 NS 5 0.3779 0.3909 NS 16.0060 18.3360 0.0285.0999 79.229 0.033*

Example 10 Field Trails Under Nitrogen Stress and Normal NitrogenConditions to Determine Grain Yield, Flowering Time, and Staygreen ofEvents Containing the NT Transgene

Corn hybrids containing the transgene were planted in the field undernitrogen stress and normal nitrogen conditions at two locations. Undernormal nitrogen, a total of 250 lbs nitrogen was applied in the form ofurea ammonium nitrate (UAN). Nitrogen stress was achieved throughdepletion of soil nitrogen reserves by planting corn with no addednitrogen for two years. Soil nitrate reserves were monitored to assessthe level of depletion. To achieve the target level of stress, UAN wasapplied by fertigation or sidedress between V2 and VT, for a total of50-150 lbs nitrogen.

Events from the construct were nested together with the null to minimizethe spatial effects of field variation; 6 reps were planted in lownitrogen, 4 reps in normal nitrogen. The grain yield of eventscontaining the transgene was compared to the yield of a transgenic null.Flowering time and staygreen were also monitored. Statistical analysiswas conducted to assess whether there is a significant improvement inyield compared with the transgenic null, taking into account row andcolumn spatial effects.

The relative yield data to nulls of PHP32100 from three locations aresummarized in FIG. 1 (under NN) and FIG. 2 (under LN).

Example 11 Screen of Candidate Genes Under Nitrogen Limiting Conditions(Prophetic)

Transgenic seed selected by the presence of selectable marker can alsobe screened for their tolerance to grow under nitrogen limitingconditions. Transgenic individuals expressing the NTs described hereinare plated on Low N medium (0.5×N-Free Hoagland's, 0.4 mM potassiumnitrate, 0.1% sucrose, 1 mM MES and 0.25% Phytagel™), such that 32transgenic individuals are grown next to 32 wild-type individuals on oneplate. Plants are evaluated at 10, 11, 12 and 13 days. If a line shows astatistically significant difference from the controls, the line isconsidered a validated nitrogen-deficiency tolerant line. After maskingthe plate image to remove background color, two different measurementsare collected for each individual: total rosetta area, and thepercentage of color that falls into a green color bin. Using hue,saturation and intensity data (HIS), the green color bin consists ofhues 50-66. Total rosetta area is used as a measure of plant biomass,whereas the green color bin has been shown by dose-response studies tobe an indicator of nitrogen assimilation.

Example 12 Screens to Identify Lines with Altered Root Architecture(Prophetic)

Arabidopsis seedlings, grown under non-limiting nitrogen conditions, maybe analyzed for altered root system architecture when compared tocontrol seedlings during early development.

Transgenic NT seedlings from in-house screen are subjected to a verticalplate assay to evaluate enhanced root growth. The results are validatedusing WinRHIZO®, as described below. T2 seeds are sterilized using 50%household bleach 0.01% triton X-100 solution and plated on petri platescontaining the following medium: 0.5×N-Free Hoagland's, 60 mM KNO₃, 0.1%sucrose, 1 mM MES and 1% Phytagel™ at a density of 4 seeds/plate. Platesare kept for three days at 4° C. to stratify seeds and then heldvertically for 11 days at 22° C. light and 20° C. dark. Photoperiod is16 h; 8 h dark and average light intensity is ˜160 μmol/m²/s. Plates areplaced vertically into the eight center positions of a 10 plate rackwith the first and last position holding blank plates. The racks and theplates within a rack are rotated every other day. Two sets of picturesare taken for each plate. The first set taking place at day 14-16 whenthe primary roots for most lines are reached the bottom of the plate,the second set of pictures two days later after more lateral roots aredeveloped. The latter set of picture is usually used for data analysis.These seedlings grown on vertical plates are analyzed for root growthwith the software WinRHIZO® (Regent Instruments Inc), an image analysissystem specifically designed for root measurement. WinRHIZO® uses thecontrast in pixels to distinguish the light root from the darkerbackground. To identify the maximum amount of roots without picking upbackground, the pixel classification is 150-170 and the filter featureis used to remove objects that have a length/width ratio less then 10.0.The area on the plates analyzed is from the edge of the plant's leavesto about 1 cm from the bottom of the plate. The exact same WinRHIZO®settings and area of analysis are used to analyze all plates within abatch. The total root length score given by WinRHIZO® for a plate isdivided by the number of plants that are germinated and are grownhalfway down the plate. Eight plates for every line are grown and theirscores are averaged. This average is then compared to the average ofeight plates containing wild type seeds that are grown at the same time.

Lines with enhanced root growth characteristics are expected to lie atthe upper extreme of the root area distributions. A sliding windowapproach is used to estimate the variance in root area for a given rackwith the assumption that there could be up to two outliers in the rack.Environmental variations in various factors including growth media,temperature, and humidity can cause significant variation in rootgrowth, especially between sow dates. Therefore the lines are grouped bysow date and shelf for the data analysis. The racks in a particular sowdate/shelf group are then sorted by mean root area. Root areadistributions for sliding windows are performed by combining data for arack, r_(i), with data from the rack with the next lowest, (r_(i−1), andthe next highest mean root area, r_(i+1). The variance of the combineddistribution is then analyzed to identify outliers in r, using aGrubbs-type approach (Barnett et al., Outliers in Statistical Data, JohnWiley & Sons, 3^(rd) edition (1994).

Example 13 NUE Assay Plant Growth (Prophetic)

Seeds of Arabidopsis thaliana (control and transgenic line), ecotypeColumbia, are surface sterilized (Sánchez et al., 2002) and then platedon to Murashige and Skoog (MS) medium containing 0.8% (w/v) Bacto-Agar(Difco). Plates are incubated for 3 days in darkness at 4° C. to breakdormancy (stratification) and transferred thereafter to growth chambers(Conviron, Manitoba, Canada) at a temperature of 20° C. under a 16-hlight/8-h dark cycle. The average light intensity is 120 μE/m2/s.Seedling are grown for 12 days and the transfer to soil based pots.Potted plants are grown on a nutrient-free soil LB2 Metro-Mix 200(Scott's Sierra Horticultural Products, Marysville, Ohio, USA) inindividual 1.5-in pots (Arabidopsis system; Lehle Seeds, Round Rock,Tex., USA) in growth chambers, as described above. Plants are wateredwith 0.6 or 6.5 mM potassium nitrate in the nutrient solution based onMurashige and Skoog (MS free Nitrogen) medium. The relative humidity ismaintained around 70%. 16-18 days later plant shoots are collected forevaluation of biomass and SPAD readings. Plants that improve NUE mayhave increased biomass at either high or low nitrate concentrations.

Example 14 Sucrose Growth Assay (Prophetic)

The Columbia line of Arabidopsis thaliana is obtained from theArabidopsis Biological Resource Center (Columbus, Ohio). For earlyanalysis (Columbia and T3 transgenic lines), seed are surface-sterilizedwith 70% ethanol for 5 min followed by 40% Clorox for 5 min and rinsedwith sterile deionized water. Surface-sterilized seed are sown ontosquare Petri plates (25 cm) containing 95 mL of sterile mediumconsisting of 0.5 Murashige and Skoog (1962) salts (Life Technologies)and 4% (w/v) phytagel (Sigma). The medium contained no supplementalsucrose. Sucrose is added to medium in 0.1%, 0.5% and 1.5%concentration. Plates are arranged vertically in plastic racks andplaced in a cold room for 3 days at 4° C. to synchronize germination.Racks with cold stratified seed are then transferred into growthchambers (Conviron, Manitoba, Canada) with day and night temperatures of22 and 20° C., respectively. The average light intensity at the level ofthe rosette is maintained at 110 mol/m2/sec1 during a 16-hr light cycledevelopment beginning at removal from the cold room (day 3 after sowing)until the seedlings are harvested on day 14. Images are taken and totalfresh weight of root and shoot are measured. Two experiments will beperformed. If expression or overexpression of an NT polynucleotidedescribed herein alters the carbon and nitrogen balance, then data mayshow that the NT polynucleotide overexpression transgenic plants areincreased or decreased root biomass and/or leaf biomass at differentsucrose concentrations when compared to wild-type Arabidopsis.

Example 15 Transformation of Gaspe Flint Derived Maize Lines with NTPolynucleotides Described Herein

Maize plants can be transformed as described in Example 4-6 andoverexpressing the NTs, for example using the ones described herein, inorder to examine the resulting phenotype. Promoters including but notlimited to the tubulin promoter (pTUB); maize ubiquitin promoter (ZMUBI), maize root metallothionein promoter (ZM-RM2); lipid transferprotein 2 promoter (LTP2); banana streak virus promoter truncatedversion promoter (BSV(TR)), maize NAS2 promoter (ZM-NAS2), and bananastreak virus promoter full version promoter (BAV (FL) and others areuseful for directing expression of the NTs in maize. Furthermore, avariety of terminators, such as, but not limited to the PINIIterminator, can be used to achieve expression of the gene of interest inGaspe Flint Derived Maize Lines.

Recipient Plants

Recipient plant cells can be from a uniform maize line having a shortlife cycle (“fast cycling”), a reduced size, and high transformationpotential. Typical of these plant cells for maize are plant cells fromany of the publicly available Gaspe Flint (GF) line varieties. Onepossible candidate plant line variety is the F1 hybrid of GF×QTM (QuickTurnaround Maize, a publicly available form of Gaspe Flint selected forgrowth under greenhouse conditions) disclosed in Tomes et al. U.S.Patent Application Publication No. 2003/0221212. Transgenic plantsobtained from this line are of such a reduced size that they can begrown in four inch pots (¼ the space needed for a normal sized maizeplant) and mature in less than 2.5 months. (Traditionally 3.5 months isrequired to obtain transgenic T0 seed once the transgenic plants areacclimated to the greenhouse.) Another suitable line is a double haploidline of GS3 (a highly transformable line) X Gaspe Flint. Yet anothersuitable line is a transformable elite inbred line carrying a transgenewhich causes early flowering, reduced stature, or both.

Transformation Protocol

Any suitable method may be used to introduce the transgenes into themaize cells, including but not limited to inoculation type proceduresusing Agrobacterium based vectors as described in Example 3.Transformation may be performed on immature embryos of the recipient(target) plant.

Precision Growth and Plant Tracking

The event population of transgenic (T0) plants resulting from thetransformed maize embryos is grown in a controlled greenhouseenvironment using a modified randomized block design to reduce oreliminate environmental error. A randomized block design is a plantlayout in which the experimental plants are divided into groups (e.g.,thirty plants per group), referred to as blocks, and each plant israndomly assigned a location with the block.

For a group of thirty plants, twenty-four transformed, experimentalplants and six control plants (plants with a set phenotype)(collectively, a “replicate group”) are placed in pots which arearranged in an array (a.k.a. a replicate group or block) on a tablelocated inside a greenhouse. Each plant, control or experimental, israndomly assigned to a location with the block which is mapped to aunique, physical greenhouse location as well as to the replicate group.Multiple replicate groups of thirty plants each may be grown in the samegreenhouse in a single experiment. The layout (arrangement) of thereplicate groups should be determined to minimize space requirements aswell as environmental effects within the greenhouse. Such a layout maybe referred to as a compressed greenhouse layout.

An alternative to the addition of a specific control group is toidentify those transgenic plants that do not express the gene ofinterest. A variety of techniques such as RT-PCR can be applied toquantitatively assess the expression level of the introduced gene. T0plants that do not express the transgene can be compared to those whichdo.

Each plant in the event population is identified and tracked throughoutthe evaluation process, and the data gathered from that plant isautomatically associated with that plant so that the gathered data canbe associated with the transgene carried by the plant. For example, eachplant container can have a machine readable label (such as a UniversalProduct Code (UPC) bar code) which includes information about the plantidentity, which in turn is correlated to a greenhouse location so thatdata obtained from the plant can be automatically associated with thatplant.

Alternatively any efficient, machine readable, plant identificationsystem can be used, such as two-dimensional matrix codes or even radiofrequency identification tags (RFID) in which the data is received andinterpreted by a radio frequency receiver/processor. See U.S. PublishedPatent Application No. 2004/0122592, incorporated herein by reference.

Phenotypic Analysis Using Three-Dimensional Imaging

Each greenhouse plant in the T0 event population, including any controlplants, is analyzed for agronomic characteristics of interest, and theagronomic data for each plant is recorded or stored in a manner so thatit is associated with the identifying data (see above) for that plant.Confirmation of a phenotype (gene effect) can be accomplished in the T1generation with a similar experimental design to that described above.

The T0 plants are analyzed at the phenotypic level using quantitative,non-destructive imaging technology throughout the plant's entiregreenhouse life cycle to assess the traits of interest. Preferably, adigital imaging analyzer is used for automatic multi-dimensionalanalyzing of total plants. The imaging may be done inside thegreenhouse. Two camera systems, located at the top and side, and anapparatus to rotate the plant, are used to view and image plants fromall sides. Images are acquired from the top, front and side of eachplant. All three images together provide sufficient information toevaluate the biomass, size and morphology of each plant.

Due to the change in size of the plants from the time the first leafappears from the soil to the time the plants are at the end of theirdevelopment, the early stages of plant development are best documentedwith a higher magnification from the top. This may be accomplished byusing a motorized zoom lens system that is fully controlled by theimaging software.

In a single imaging analysis operation, the following events occur: (1)the plant is conveyed inside the analyzer area, rotated 360 degrees soits machine readable label can be read, and left at rest until itsleaves stop moving; (2) the side image is taken and entered into adatabase; (3) the plant is rotated 90 degrees and again left at restuntil its leaves stop moving, and (4) the plant is transported out ofthe analyzer.

Plants are allowed at least six hours of darkness per twenty four hourperiod in order to have a normal day/night cycle.

Imaging Instrumentation

Any suitable imaging instrumentation may be used, including but notlimited to light spectrum digital imaging instrumentation commerciallyavailable from LemnaTec GmbH of Wurselen, Germany. The images are takenand analyzed with a LemnaTec Scanalyzer HTS LT-0001-2 having a ½″ ITProgressive Scan IEE CCD imaging device. The imaging cameras may beequipped with a motor zoom, motor aperture and motor focus. All camerasettings may be made using LemnaTec software. Preferably, theinstrumental variance of the imaging analyzer is less than about 5% formajor components and less than about 10% for minor components.

Software

The imaging analysis system comprises a LemnaTec HTS Bonit softwareprogram for color and architecture analysis and a server database forstoring data from about 500,000 analyses, including the analysis dates.The original images and the analyzed images are stored together to allowthe user to do as much reanalyzing as desired. The database can beconnected to the imaging hardware for automatic data collection andstorage. A variety of commercially available software systems (e.g.Matlab, others) can be used for quantitative interpretation of theimaging data, and any of these software systems can be applied to theimage data set.

Conveyor System

A conveyor system with a plant rotating device may be used to transportthe plants to the imaging area and rotate them during imaging. Forexample, up to four plants, each with a maximum height of 1.5 m, areloaded onto cars that travel over the circulating conveyor system andthrough the imaging measurement area. In this case the total footprintof the unit (imaging analyzer and conveyor loop) is about 5 m×5 m.

The conveyor system can be enlarged to accommodate more plants at atime. The plants are transported along the conveyor loop to the imagingarea and are analyzed for up to 50 seconds per plant. Three views of theplant are taken. The conveyor system, as well as the imaging equipment,should be capable of being used in greenhouse environmental conditions.

Illumination

Any suitable mode of illumination may be used for the image acquisition.For example, a top light above a black background can be used.Alternatively, a combination of top- and backlight using a whitebackground can be used. The illuminated area should be housed to ensureconstant illumination conditions. The housing should be longer than themeasurement area so that constant light conditions prevail withoutrequiring the opening and closing or doors. Alternatively, theillumination can be varied to cause excitation of either transgene(e.g., green fluorescent protein (GFP), red fluorescent protein (RFP))or endogenous (e.g. Chlorophyll) fluorophores.

Biomass Estimation Based on Three-Dimensional Imaging

For best estimation of biomass the plant images should be taken from atleast three axes, preferably the top and two side (sides 1 and 2) views.These images are then analyzed to separate the plant from thebackground, pot and pollen control bag (if applicable). The volume ofthe plant can be estimated by the calculation:Volume(voxels)=√{square root over (TopArea(pixels))}×√{square root over(Side1Area(pixels))}×√{square root over (Side2Area(pixels))}

In the equation above the units of volume and area are “arbitraryunits”. Arbitrary units are entirely sufficient to detect gene effectson plant size and growth in this system because what is desired is todetect differences (both positive-larger and negative-smaller) from theexperimental mean, or control mean. The arbitrary units of size (e.g.area) may be trivially converted to physical measurements by theaddition of a physical reference to the imaging process. For instance, aphysical reference of known area can be included in both top and sideimaging processes. Based on the area of these physical references aconversion factor can be determined to allow conversion from pixels to aunit of area such as square centimeters (cm²). The physical referencemay or may not be an independent sample. For instance, the pot, with aknown diameter and height, could serve as an adequate physicalreference.

Color Classification

The imaging technology may also be used to determine plant color and toassign plant colors to various color classes. The assignment of imagecolors to color classes is an inherent feature of the LemnaTec software.With other image analysis software systems color classification may bedetermined by a variety of computational approaches.

For the determination of plant size and growth parameters, a usefulclassification scheme is to define a simple color scheme including twoor three shades of green and, in addition, a color class for chlorosis,necrosis and bleaching, should these conditions occur. A backgroundcolor class which includes non plant colors in the image (for examplepot and soil colors) is also used and these pixels are specificallyexcluded from the determination of size. The plants are analyzed undercontrolled constant illumination so that any change within one plantover time, or between plants or different batches of plants (e.g.seasonal differences) can be quantified.

In addition to its usefulness in determining plant size growth, colorclassification can be used to assess other yield component traits. Forthese other yield component traits additional color classificationschemes may be used. For instance, the trait known as “staygreen”, whichhas been associated with improvements in yield, may be assessed by acolor classification that separates shades of green from shades ofyellow and brown (which are indicative of senescing tissues). Byapplying this color classification to images taken toward the end of theT0 or T1 plants' life cycle, plants that have increased amounts of greencolors relative to yellow and brown colors (expressed, for instance, asGreen/Yellow Ratio) may be identified. Plants with a significantdifference in this Green/Yellow ratio can be identified as carryingtransgenes which impact this important agronomic trait.

The skilled plant biologist will recognize that other plant colors arisewhich can indicate plant health or stress response (for instanceanthocyanins), and that other color classification schemes can providefurther measures of gene action in traits related to these responses.

Plant Architecture Analysis

Transgenes which modify plant architecture parameters may also beidentified using the present invention, including such parameters asmaximum height and width, internodal distances, angle between leaves andstem, number of leaves starting at nodes and leaf length. The LemnaTecsystem software may be used to determine plant architecture as follows.The plant is reduced to its main geometric architecture in a firstimaging step and then, based on this image, parameterized identificationof the different architecture parameters can be performed. Transgenesthat modify any of these architecture parameters either singly or incombination can be identified by applying the statistical approachespreviously described.

Pollen Shed Date

Pollen shed date is an important parameter to be analyzed in atransformed plant, and may be determined by the first appearance on theplant of an active male flower. To find the male flower object, theupper end of the stem is classified by color to detect yellow or violetanthers. This color classification analysis is then used to define anactive flower, which in turn can be used to calculate pollen shed date.

Alternatively, pollen shed date and other easily visually detected plantattributes (e.g. pollination date, first silk date) can be recorded bythe personnel responsible for performing plant care. To maximize dataintegrity and process efficiency this data is tracked by utilizing thesame barcodes utilized by the LemnaTec light spectrum digital analyzingdevice. A computer with a barcode reader, a palm device, or a notebookPC may be used for ease of data capture recording time of observation,plant identifier, and the operator who captured the data.

Orientation of the Plants

Mature maize plants grown at densities approximating commercial plantingoften have a planar architecture. That is, the plant has a clearlydiscernable broad side, and a narrow side. The image of the plant fromthe broadside is determined. To each plant a well defined basicorientation is assigned to obtain the maximum difference between thebroadside and edgewise images. The top image is used to determine themain axis of the plant, and an additional rotating device is used toturn the plant to the appropriate orientation prior to starting the mainimage acquisition.

Example 16 Transgenic Maize Plants (Prophetic)

T₀ transgenic maize plants containing the NT construct under the controlof a promoter were generated. These plants were grown in greenhouseconditions, under the FASTCORN system, as detailed in U.S. PatentApplication Publication 2003/0221212, U.S. patent application Ser. No.10/367,417.

Each of the plants was analyzed for measurable alteration in one or moreof the following characteristics in the following manner.

T₁ progeny derived from self fertilization each T₀ plant containing asingle copy of each NT construct that were found to segregate 1:1 forthe transgenic event were analyzed for improved growth rate in low KNO₃.Growth was monitored up to anthesis when cumulative plant growth, growthrate and ear weight were determined for transgene positive, transgenenull, and non-transformed controls events. The distribution of thephenotype of individual plants was compared to the distribution of acontrol set and to the distribution of all the remaining treatments.Variances for each set were calculated and compared using an F test,comparing the event variance to a non-transgenic control set varianceand to the pooled variance of the remaining events in the experiment.The greater the response to KNO₃, the greater the variance within anevent set and the greater the F value. Positive results will be comparedto the distribution of the transgene within the event to make sure theresponse segregates with the transgene.

Example 17 Transgenic Event Analysis from Field Plots (Prophetic)

Transgenic events are evaluated in field plots where yield is limited byreducing fertilizer application by 30% or more. Improvements in yield,yield components, or other agronomic traits between transgenic andnon-transgenic plants in these reduced nitrogen fertility plots are usedto assess improvements in nitrogen utilization contributed by expressionof transgenic events. Similar comparisons are made in plots supplementedwith recommended nitrogen fertility rates. Effective transgenic eventsare those that achieve similar yields in the nitrogen-limited and normalnitrogen experiments.

Example 18 Assays to Determine Alterations of Root Architecture in Maize

Transgenic maize plants are assayed for changes in root architecture atseedling stage, flowering time or maturity. Assays to measurealterations of root architecture of maize plants include, but are notlimited to the methods outlined below. To facilitate manual or automatedassays of root architecture alterations, corn plants can be grown inclear pots.

-   -   1) Root mass (dry weights). Plants are grown in Turface, a        growth media that allows easy separation of roots. Oven-dried        shoot and root tissues are weighed and a root/shoot ratio        calculated.    -   2) Levels of lateral root branching. The extent of lateral root        branching (e.g. lateral root number, lateral root length) is        determined by sub-sampling a complete root system, imaging with        a flat-bed scanner or a digital camera and analyzing with        WinRHIZO™ software (Regent Instruments Inc.).    -   3) Root band width measurements. The root band is the band or        mass of roots that forms at the bottom of greenhouse pots as the        plants mature. The thickness of the root band is measured in mm        at maturity as a rough estimate of root mass.    -   4) Nodal root count. The number of crown roots coming off the        upper nodes can be determined after separating the root from the        support medium (e.g. potting mix). In addition the angle of        crown roots and/or brace roots can be measured. Digital analysis        of the nodal roots and amount of branching of nodal roots form        another extension to the aforementioned manual method.

All data taken on root phenotype are subjected to statistical analysis,normally a t-test to compare the transgenic roots with that ofnon-transgenic sibling plants. One-way ANOVA may also be used in caseswhere multiple events and/or constructs are involved in the analysis.

Example 19 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing an antisense NTsequences operably linked to an ubiquitin promoter as follows. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected fromsurface-sterilized, immature seeds of the soybean cultivar A2872, arecultured in the light or dark at 26° C. on an appropriate agar mediumfor six to ten weeks. Somatic embryos producing secondary embryos arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiplied as early,globular-staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising an NT sequence describedherein operably linked to the ubiquitin promoter can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 20 Sunflower Meristem Tissue Transformation (Prophetic)

Sunflower meristem tissues are transformed with an expression cassettecontaining an NT sequence described herein operably linked to aubiquitin promoter as follows (see also, European Patent Number EP 0486233, herein incorporated by reference, and Malone-Schoneberg, et al.,(1994) Plant Science 103:199-207). Mature sunflower seed (Helianthusannuus L.) are dehulled using a single wheat-head thresher. Seeds aresurface sterilized for 30 minutes in a 20% Clorox bleach solution withthe addition of two drops of Tween 20 per 50 ml of solution. The seedsare rinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer, et al. (Schrammeijer, et al.,(1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled waterfor 60 minutes following the surface sterilization procedure. Thecotyledons of each seed are then broken off, producing a clean fractureat the plane of the embryonic axis. Following excision of the root tip,the explants are bisected longitudinally between the primordial leaves.The two halves are placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., (1962) Physiol.Plant., 15:473-497), Shepard's vitamin additions (Shepard, (1980) inEmergent Techniques for the Genetic Improvement of Crops (University ofMinnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/lsucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-aceticacid (IAA), 0.1 mg/l gibberellic acid (GA₃), pH 5.6, and 8 g/l Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS1000® particle accelerationdevice.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the NT polynucletide operably linkedto the ubiquitin promoter is introduced into Agrobacterium strain EHA105via freeze-thawing as described by Holsters, et al., (1978) Mol. Gen.Genet. 163:181-187. This plasmid further comprises a kanamycinselectable marker gene (i.e, nptII). Bacteria for plant transformationexperiments are grown overnight (28° C. and 100 RPM continuousagitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/lBactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriate antibioticsrequired for bacterial strain and binary plasmid maintenance. Thesuspension is used when it reaches an OD₆₀₀ of about 0.4 to 0.8. TheAgrobacterium cells are pelleted and resuspended at a final OD₆₀₀ of 0.5in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl,and 0.3 gm/l MgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for a modulation in meristemdevelopment (i.e., an alteration of size and appearance of shoot andfloral meristems).

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%gelrite, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl, and the transformed shoot inserted intothe cut. The entire area is wrapped with parafilm to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by nitrate uptake, NT orNUE activity analysis of leaf extracts while transgenic seeds harvestedfrom NPTII-positive T₀ plants are identified by nitrate uptake, NT orNUE activity analysis of small portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Cloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagarat pH 5.6) for 24 hours under the dark. The primary leaves are removedto expose the apical meristem, around 40 explants are placed with theapical dome facing upward in a 2 cm circle in the center of 374M (GBAmedium with 1.2% Phytagar), and then cultured on the medium for 24 hoursin the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD 600.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for a modulation in meristem development (i.e., an alterationof size and appearance of shoot and floral meristems). After positiveexplants are identified, those shoots that fail to exhibit modified NTactivity are discarded, and every positive explant is subdivided intonodal explants. One nodal explant contains at least one potential node.The nodal segments are cultured on GBA medium for three to four days topromote the formation of auxiliary buds from each node. Then they aretransferred to 374C medium and allowed to develop for an additional fourweeks. Developing buds are separated and cultured for an additional fourweeks on 374C medium. Pooled leaf samples from each newly recoveredshoot are screened again by the appropriate protein activity assay. Atthis time, the positive shoots recovered from a single node willgenerally have been enriched in the transgenic sector detected in theinitial assay prior to nodal culture.

Recovered shoots positive for modified NT expression are grafted toPioneer hybrid 6440 in vitro-grown sunflower seedling rootstock. Therootstocks are prepared in the following manner. Seeds are dehulled andsurface-sterilized for 20 minutes in a 20% Clorox bleach solution withthe addition of two to three drops of Tween 20 per 100 ml of solution,and are rinsed three times with distilled water. The sterilized seedsare germinated on the filter moistened with water for three days, thenthey are transferred into 48 medium (half-strength MS salt, 0.5%sucrose, 0.3% gelrite pH 5.0) and grown at 26° C. under the dark forthree days, then incubated at 16-hour-day culture conditions. The upperportion of selected seedling is removed, a vertical slice is made ineach hypocotyl, and a transformed shoot is inserted into a V-cut. Thecut area is wrapped with parafilm. After one week of culture on themedium, grafted plants are transferred to soil. In the first two weeks,they are maintained under high humidity conditions to acclimatize to agreenhouse environment.

Example 21 Rice Tissue Transformation (Prophetic)

One method for transforming DNA into cells of higher plants that isavailable to those skilled in the art is high-velocity ballisticbombardment using metal particles coated with the nucleic acidconstructs of interest (see, Klein, et al., Nature (1987) (London)327:70-73, and see U.S. Pat. No. 4,945,050). A Biolistic PDS-1000/He(BioRAD Laboratories, Hercules, Calif.) is used for thesecomplementation experiments. The particle bombardment technique is usedto transform the NT mutants and wild type rice with DNA fragments

The bacterial hygromycin B phosphotransferase (Hpt II) gene fromStreptomyces hygroscopicus that confers resistance to the antibiotic isused as the selectable marker for rice transformation. In the vector,pML18, the Hpt II gene was engineered with the 35S promoter fromCauliflower Mosaic Virus and the termination and polyadenylation signalsfrom the octopine synthase gene of Agrobacterium tumefaciens. pML18 wasdescribed in WO 97/47731, which was published on Dec. 18, 1997, thedisclosure of which is hereby incorporated by reference.

Embryogenic callus cultures derived from the scutellum of germinatingrice seeds serve as source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is the transferred to CMmedia (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al.,1985, Sci. Sinica 18: 659-668). Callus cultures are maintained on CM byroutine sub-culture at two week intervals and used for transformationwithin 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28° C. for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr inthe dark. The petri dish lids are then left ajar for 20-45 minutes in asterile hood to allow moisture on tissue to dissipate.

Each genomic DNA fragment is co-precipitated with pML18 containing theselectable marker for rice transformation onto the surface of goldparticles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio oftrait:selectable marker DNAs are added to 50 μl aliquot of goldparticles that have been resuspended at a concentration of 60 mg ml⁻¹.Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a0.1 M solution) are then added to the gold-DNA suspension as the tube isvortexing for 3 min. The gold particles are centrifuged in a microfugefor 1 sec and the supernatant removed. The gold particles are thenwashed twice with 1 ml of absolute ethanol and then resuspended in 50 μlof absolute ethanol and sonicated (bath sonicator) for one second todisperse the gold particles. The gold suspension is incubated at −70° C.for five minutes and sonicated (bath sonicator) if needed to dispersethe particles. Six μl of the DNA-coated gold particles are then loadedonto mylar macrocarrier disks and the ethanol is allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 psi. Thetissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Two to four plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates are incubated in the dark for 4 weeks at 27-28° C. After 4weeks, transgenic callus events are identified, transferred to fresh SMplates and grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite+50 ppm hyg B) for 2weeks in the dark at 25° C. After 2 weeks the callus is transferred toRM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4%gelrite+50 ppm hyg B) and placed under cool white light (˜40 μEm⁻²s⁻¹)with a 12 hr photo period at 25° C. and 30-40% humidity. After 2-4 weeksin the light, callus begin to organize, and form shoots. Shoots areremoved from surrounding callus/media and gently transferred to RM3media (½×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppmhygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, Mo.) andincubation is continued using the same conditions as described in theprevious step.

Plants are transferred from RM3 to 4″ pots containing Metro mix 350after 2-3 weeks, when sufficient root and shoot growth have occurred.The seed obtained from the transgenic plants is examined for geneticcomplementation of the NT mutation with the wild-type genomic DNAcontaining the NT polynucleotide.

Example 22 NUE Assay

Using the protocol detailed in U.S. Patent Application Ser. No.61/227,276, a triphenyltetrazolium chloride (TTC) assay may be performedto evaluate genes for NUE, for example, increased transport activity inroots, using transgenic maize lines.

Example 23 Variants of NT Sequences (Prophetic)

A. Variant Nucleotide Sequences of NT Proteins that do not Alter theEncoded Amino Acid Sequence

The NT nucleotide sequences described herein are used to generatevariant nucleotide sequences having the nucleotide sequence of the openreading frame with about 70%, 75%, 80%, 85%, 90%, and 95% nucleotidesequence identity when compared to the starting unaltered ORF nucleotidesequence of the corresponding SEQ ID NO. These functional variants aregenerated using a standard codon table. While the nucleotide sequence ofthe variants are altered, the amino acid sequence encoded by the openreading frames do not change.

B. Variant Amino Acid Sequences of NT Polypeptides

Variant amino acid sequences of the NT polypeptides are generated. Inthis example, one amino acid is altered. Specifically, the open readingframes are reviewed to determine the appropriate amino acid alteration.The selection of the amino acid to change is made by consulting theprotein alignment (with the other orthologs and other gene familymembers from various species). An amino acid is selected that is deemednot to be under high selection pressure (not highly conserved) and whichis rather easily substituted by an amino acid with similar chemicalcharacteristics (i.e., similar functional side-chain). Using the proteinalignment, an appropriate amino acid can be changed. Once the targetedamino acid is identified, the procedure outlined in the followingsection C is followed. Variants having about 70%, 75%, 80%, 85%, 90%,and 95% nucleic acid sequence identity are generated using this method.

C. Additional Variant Amino Acid Sequences of NT Polypeptides

In this example, artificial protein sequences are created having 80%,85%, 90%, and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom the alignment and then the judicious application of an amino acidsubstitutions table. These parts will be discussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among NT protein or among the otherNT polypeptides. Based on the sequence alignment, the various regions ofthe NT polypeptide that can likely be altered are represented in lowercase letters, while the conserved regions are represented by capitalletters. It is recognized that conservative substitutions can be made inthe conserved regions below without altering function. In addition, oneof skill will understand that functional variants of the NT sequence ofthe invention can have minor non-conserved amino acid alterations in theconserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95%, and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 7.

TABLE 7 Substitution Table Strongly Similar Rank of Amino and OptimalOrder to Acid Substitution Change Comment I L, V 1 50:50 substitution LI, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged is identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C, and P are not changed in any circumstance. The changes will occurwith isoleucine first, sweeping N-terminal to C-terminal. Then leucine,and so on down the list until the desired target it reached. Interimnumber substitutions can be made so as not to cause reversal of changes.The list is ordered 1-17, so start with as many isoleucine changes asneeded before leucine, and so on down to methionine. Clearly many aminoacids will in this manner not need to be changed. L, I and V willinvolve a 50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof the NT polypeptides are generating having about 80%, 85%, 90%, and95% amino acid identity to the starting unaltered ORF nucleotidesequence of SEQ ID NO:1 or 3.

Example 24 Nitrate Uptake Assay to Determining Expression Level andActivity of YNT1 MO as Compared to Wild Type YNT1 in Maize

Corn seeds containing the transgene are planted in small pots and waterwith nutrient solution containing nitrate as the sole nitrogen source.The pots with seedlings are transferred into a larger containercontaining the same nutrient solution. An aliquot of nutrient solutionis removed and the amount of nitrate is determined. Two weeks aftertransfer the plants are harvested for biomass and total reduced nitrogenmeasurements. The loss of nitrate from the nutrient solution is used todetermine the nitrate uptake.

Example 25 Maize Transient Expression Assay to Determine ProteinExpression Level (Prophetic)

Protein expression level can be determined by Agrobacterium-mediatedtransient expression assay in maize. A binary plasmid vector comprisingthe expression cassette that contains the NT polynucletide driven bymaize ubiquitin promoter or PEPC promoter is introduced intoAgrobacterium strain LBA4404 via electroporation as that described inExample 6. Maize seedlings, young leaf tissues, or suspension cells areinfected by the Agrobacterium culture carrying the NT expressioncassette by infiltration or vacuum. The infected plant materials arerecovered for few days under desired conditions, e.g. green house orgrowth camber with nutrients/medium. Proteins are extracted from theinfected tissues and analyzed by Western blot following the standardprocedure.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

That which is claimed:
 1. A recombinant nucleic acid comprising anitrate transporter (NT) encoding polynucleotide and a nitrate reductase(NR) encoding polynucleotide; wherein said NT polynucleotide is selectedfrom the group consisting of: (a) a polynucleotide having at least 99%identity to SEQ ID NO: 3, comprising a nucleotide that has beensubstituted, wherein the nucleotide substitution is one or more of thesubstitutions shown in FIG. 3, and wherein the polynucleotide encodes apolypeptide having NT activity; (b) a polynucleotide comprising thesequence set forth in SEQ ID NO: 3; and wherein the nitrate reductasepolynucleotide is selected from the group consisting of: (i) apolynucleotide that encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 5 or 9; (ii) a polynucleotidecomprising the sequence set forth in SEQ ID NO: 4 or 8; and (iii) apolynucleotide having at least 95% sequence identity to at least onenucleotide sequence selected from the group consisting of SEQ ID NOs: 4and 8, wherein the % sequence identity is based on the entire encodingregion and is determined by BLAST 2.0 under default parameters, andwherein the polynucleotide encodes a polypeptide having NR activity andcomprising the A551 G substitution, the S561 D substitution, or the A551G and S561 D substitutions.
 2. The polynucleotide of claim 1, whereinthe polynucleotide encodes a NT polypeptide that confers to a plantincreased yield enhanced plant tissue growth as indicated by one of thefollowing characteristics: enhanced root growth, increased seed size,increased shoot biomass, increased seed weight, increased embryo size,increased leaf size, increased seedling vigor, enhanced silk emergence,increased ear size or chlorophyll content, wherein the yield of theplant is compared to a control plant, and wherein the control plant doesnot contain the NT polynucleotide.
 3. A vector comprising at least onepolynucleotide, or an expression cassette comprising at least onepolynucleotide operably linked to a promoter; wherein the polynucleotideis the polynucleotide of claim
 1. 4. A host cell comprising at least oneexpression cassette of claim
 3. 5. The host cell of claim 4, wherein thehost cell is a dicotyledonous or monocotyledonous plant cell.
 6. Atransgenic plant comprising at least one expression cassette of claim 3.7. A seed produced by the transgenic plant of claim 6, wherein the seedcomprises the expression cassette.